Fifteen expressed sequence tag (EST)-derived simple sequence repeats (EST-SSRs) were used to investigate genetic diversity in 139 plants obtained from seeds of 35 watermelon accessions collected from all the geographical provinces of Zimbabwe. In addition, 15 plants representing three commercial varieties developed in the United States (USA) were analyzed for comparison. A total of 65 alleles were detected among all the watermelon accessions. For the 13 polymorphic EST-SSR loci, number of alleles per locus varied from 2 to 13, with an average of 5 alleles per locus. Values for the polymorphic information content increased as the number of alleles increased, and varied from 0.15 to 0.77 with an average of 0.54 suggesting sufficient discriminatory power. Both cluster analysis and principal coordinate analysis (PCA) produced two major clusters; one with the 22 cow-melon accessions and the other with the 16 sweet watermelon accessions. Within the sweet watermelon group, two distinct sub-clusters formed, one of which contained only two of the commercial varieties from USA. Partitioning of genetic variation in the Zimbabwean material using analysis of molecular variation (AMOVA) revealed that 64% of the total variation resides between the two major forms, i.e. sweet watermelons and cow-melons, 28% between accessions within forms and 8% within accessions. The EST-SSR markers revealed a somewhat higher diversity in sweet watermelon accessions compared to that of cow-melons. This finding is contrary to previous reports using other markers (genomic SSR loci or RAPD) and/or a plant material that is likely to have experienced more stringent selection procedures compared to the landraces analyzed in our study.