Technical Abstract: Sclerotinia trifoliorum was recently found to infect chickpea (Cicer arientinum) in North America. Attempts to study the population biology of this pathogen using previously developed microsatellite markers for closely related species S. sclerotiorum and S. sub-arctica resulted in little or no amplification or low levels of polymorphism. This study reports the development, characterization, transferability and utility of thirty-three microsatellite loci developed from a microsatellite-enriched library of S. trifoliorum. An average of 6.5 alleles per locus (range 3-12) and a mean expected heterozygosity of 0.63 (range 0.26-0.9) was found using a panel of 42 isolates of S. trifoliorum. Based on the genome sequences of S. sclerotiorum, the microsatellite loci are dispersed throughout the genome. However, 50% (265 of the 528) of pairwise comparisons of the 33 microsatellite loci had significant linkage disequilibrium. This was not unexpected since S. trifoliorum is a partially outcrossing fungus. Thirty of the 33 loci were successfully applied to congeneric S. sclerotiorum and 28 loci were polymorphic, ten loci in S. minor and one loci in S. homoeocarpa. These markers are therefore useful for population structure assessment, QTL mapping and ecological analyses in S. trifoliorum and potentially in other Sclerotinia species.[National Sclerotinia Initiative]