Microsatellite markers or SSR (Simple Sequence Repeats) have proved to be an excellent tool for cultivar identification, pedigree analysis and the evaluation of genetic distance among organisms. Soybean cultivars have been characterized mainly by morphological and biochemical traits. However, these traits have not been sufficient to characterize the large number of cultivars eligible to receive protection under the Brazilian Cultivar Protection Act. In order to define new soybean cultivar markers, the alleles of twelve SSR loci of 186 Brazilian soybean cultivars were studied by estimating the variation in their size range and their respective frequencies. On average, 5.3 alleles per locus were detected, with a mean genetic diversity of 0.64 ± 0.12. These loci were used to distinguish morphologically similar groups, presenting a mean similarity coefficient of 0.46; their use allowed to determine 184 profiles for the 186 cultivars. A dendrogram based on the SSR loci profiles showed good agreement with the cultivar pedigree information.