Calopogonium mucunoides Desv. is a species native of South and Central America that is used as green manure and a pasture crop. The molecular genetic diversity was characterized in 195 C. mucunoides accessions from a germplasm collection using 17 microsatellite markers. Outcrossing rate was estimated after the evaluation of six microsatellite loci in 200 genotypes originated from 10 open-pollinated progenies (20 genotypes per progeny). Six genetic clusters were identified in the germplasm collection by the STRUCTURE software analysis, neighbor-joining tree comparisons and principal component analysis, which highly correlated with the geographic locations where these accessions were originated or collected. These results were confirmed using AMOVA. The largest portion of the genetic variation was observed among clusters (64.38%). The results indicated that: multilocus outcrossing rate (t m) was 16.3%, suggesting a mixed mating system with a predominance of autogamy; single locus outcrossing rate (t s) was 11%; difference (t m −t s) was 0.054, indicating that only 5.4% of outcrossing occurred among related individuals; paternity correlation (r p) was 33% suggesting a low probability of finding full sibs among the progeny; parental coefficient of inbreeding (F m) was 5.0%, indicating a low degree of inbreeding in each parent. A core collection for C. mucunoides was assembled to capture the allelic diversity found in this study. The complete allelic diversity was represented by only 15 accessions. These results should be useful for exploiting the genetic resources of C. mucunoides and could influence future conservation efforts and breeding programs.