Sclerotinia trifoliorum is an important pathogen of forage legumes and some grain legumes. Attempts to study its population biology using microsatellite markers developed for Sclerotinia sclerotiorum and Sclerotinia subarctica resulted in no amplification or low levels of polymorphism. This study reports the development and characterization of 33 microsatellite loci developed from a microsatellite-enriched library of S. trifoliorum. Based on a population of 42 isolates of S. trifoliorum, these microsatellite markers are highly polymorphic, with a mean of 6.5 alleles per locus (range 3-12) and a mean expected heterozygosity of 0.63 (range 0.26-0.9). Based on locations of these marker sequences in the S. sclerotiorum genome, these microsatellite loci are dispersed throughout the genome. However, 50% (265 of 528) of pairwise comparisons of the 33 microsatellite loci had significant linkage disequilibrium, which could be explained by the mixed mating systems (homothallism and heterothallism) and clonal reproduction of S. trifoliorum. Thirty of the 33 loci were successfully applied to S. sclerotiorum, and 28 loci were polymorphic. However, only 10 loci are applicable to Sclerotinia minor and 1 locus to Sclerotinia homoeocarpa. These markers are therefore useful for population structure assessment, QTL mapping, and ecological analyses in S. trifoliorum and potentially in other Sclerotinia species.