Abstract Cryptocoryne cordata var. cordata (2n = 34) is an aquatic plant species distributed from the southern part of Peninsular Thailand through the Malay Peninsula. It propagates both sexually and asexually via stolons. The current study is aimed at developing nuclear microsatellite markers for the species using next generation sequencing (Roche 454 pyrosequencing) from genomic DNA. A total of 41,653 reads was generated, of which 3636 fragments contained at least one repeat motif. Seventy two primer sets in the flanking region of dinucleotide, trinucleotide and tetranucleotides repeat motifs were designed and tested for efficiency in polymerase chain reaction amplification. Using these primer sets, 11 new microsatellite marker loci were successfully amplified with unambiguous polymorphic alleles exhibited across 30 individuals examined. The number of alleles per locus ranged from 2 to 6, while observed and expected heterozygosity ranged from 0.8190 to 1.0000 and 0.5401 to 0.7548, respectively. Genotype frequencies at all loci departed significantly from Hardy–Weinberg equilibrium. Linkage disequilibrium was not detected between any pair of loci. Cross-species amplification was successful across a panel of ten Cryptocoryne species. The markers described in this study will be useful for evaluating genetic diversity within and between populations, levels of gene flow, and the population dynamics of clones. They will be of further value in the development of effective conservation programs for Cryptocoryne species.