Inherent intraspecific genetic variability is a ubiquitous trait among microbial pathogens, resulting in isolates that exhibit unique genetic characteristics influencing their pathogenicity in different environmental contexts. Metarhizium is a genus of entomopathogenic fungi that displays notable variation between species in terms of genetic content and host range. Due to their ability to regulate insect populations, several species of Metarhizium have been recognized for their potential as effective biological control agents against insect pests in agroecosystems. Despite this, the naturally occurring intraspecific variation in genetic or phenotypic contexts has received limited attention. Given the widespread distribution of many Metarhizium species and their diverse host populations, there is likely cryptic genotypic and phenotypic within-species diversity in globally distributed isolates. Comprehensive knowledge of intraspecific variation is essential for elucidating the adaptive potential and evolutionary mechanisms of fungal pathogens. Furthermore, the identification of natural variation in isolates can enable researchers to select the most suitable candidates for biological control of specific insect pests, leading to more effective and sustainable pest management strategies. The potential applications of Metarhizium in biological control are vast, and the exploration of its intraspecific variability can provide insights into the ecology and evolution of fungal pathogens. This thesis focuses on studying the variation within M. acridum, a species that exclusively infects orthopteran insects. Although M. acridum can be found in different regions of the world, its distribution is limited to sub-tropical and tropical areas, where its orthopteran hosts are present. While M. acridum has evolved physical and genetic characteristics distinct from its sister taxa, the intraspecific diversity of this taxon is still largely unknown. The three manuscripts presented in this thesis provide a comprehensive characterization of this variation from multiple perspectives. Chapter 2 of this thesis delves into the examination of phenotypic and genotypic variation, of nine M. acridum isolates. In terms of phenotypic variation, this study focuses on (1) virulence on two locust species and (2) growth rates on diverse nutrient sources. The results reveal significant variation in virulence and growth, indicative of pathogenic divergence and the potential for ecological specialization. Moreover, through the utilization of single-nucleotide polymorphisms (SNPs), this investigation sheds light on the considerable diversity present in subtilisin-like proteases belonging to the Pr1 family of M. acridum, and presents compelling evidence of gene duplication events in certain isolates, thereby offering novel insights into the candidate evolutionary forces driving intraspecific diversity in this fungal species. In Chapter 3 of this thesis, we conducted an extensive analysis of the genomic diversity of the specialist fungal entomopathogen, M. acridum. Using a gene-centric pangenome approach and examining seven complete genomes, we found that approx- imately 25% of the pangenome consists of accessory genes enriched in secondary metabolite production, nutrient transport, and chromosome organization. Our analysis also revealed functional compartmentalization of the genome, with the core genome enriched in carbohydrate-active enzymes and the accessory genome enriched in effectors located in gene-sparse regions, exhibiting patterns similar to other well-characterized fungal plant pathogens. Moreover, we identified the first naturally occurring accessory chromosome in M. acridum, which is enriched in functions related to sexual reproduction, offering new insights into the evolutionary mechanisms driving fungal adaptation and niche specialization. Overall, our findings represent the functional genomic diversity of M. acridum, and represent the first pangenome study of an entomopathogenic fungus to date. For Chapter 4, the study focuses on examining the initial mechanisms involved in a host shift of the specialist pathogen, M. acridum. The present study aimed to investigate whether isolates differing in virulence and genotype, exhibit similar phenotypic plasticity in virulence when exposed to a novel host. To this end, we artificially induced a host shift environment in three selected isolates and monitored their response to serial passaging in a novel host species. The study found that initial virulence is a strong predictor of capability to infect a novel host, indicative of the role of standing genetic variation in facilitating host shifts. This study also explores change in gene expression patterns following serial passaging, showing the role of phenotypic plasticity. In this regard, several detoxification genes were down-regulated in all passaged lineages of M. acridum, and this down-regulation may facilitate infection within the novel host. Overall, the study provides new insights into the genetic mechanisms underlying the rapid adaptation of fungal pathogens to a novel host and emphasizes the role of standing genetic variation and phenotypic plasticity in facilitating the adaptation of fungal pathogens to novel or stressful host environments. In conclusion, M. acridum exhibits notable intraspecific genetic variability that presents in phenotypic characteristics related to virulence. The three manuscripts presented in this thesis comprehensively characterize this variability, revealing considerable heterogeneity in terms of pathogenicity, genomic composition, and adaptability to novel stressors. Although the underlying mechanisms driving these differences remain unclear, the results suggest the existence of cryptic population structure and variation in pathogenic capacity. Notably, the research highlights the functional genomic diversity of M. acridum, including the discovery of the first naturally occurring accessory chromosome in the genus, and mutational diversity of specific subtilisin-like proteases. Additionally, our findings demonstrate the crucial roles of standing genetic variation and phenotypic plasticity in facilitating the adaptation of M. acridum to novel or stressful host environments. In light of these results, we anticipate that our research will provide new insights into the ecology and evolution of fungal pathogens, with the potential to inform more effective and sustainable pest management strategies that exploit natural variation in M. acridum isolates.