This study describes the genetic differentiation of a collection of 134 watermelon landrace accessions from Mali, representing red fleshed dessert and white fleshed seed and cooking type watermelons from five regions, plus three commercial dessert type cultivars with red flesh. The material was represented by in total 397 plants and was analysed using 24 microsatellite primer sets, which differentiated 129 alleles across all loci. Analyses of molecular variance ascribed 51 % of the variation among the landrace accessions, and 14 % of the variation could be ascribed to the regions. Partitioning the accessions into use groups (dessert, cooking, seed processing) explained 25 % of the variation. When categorising the accessions further into 10 landrace types, differentiated on the basis of use groups, local accession name, flesh colour and seed phenotype, these landrace types explained 26 % of the variation. Analysis with the software Structure revealed that the accessions with confidence could be separated into two major genetic groups, related to flesh colour (red and white) of the watermelon fruits. The same analysis further indicated that the material may be differentiated into eight genetic sub-groups. One group included again the red fleshed dessert types with local and commercial origin, while the remaining seven genetic sub-groups comprised the white fleshed landrace types used for seed processing and cooking, as well as white fleshed types of one dessert type. Some of the seed and cooking types were to a large extent different and assigned to one genetic sub-group each. A Mantel test, based on geographical and genetic distance matrices showed a positive correlation, indicating that seed exchange has not overcome local adaptation.