There are numerous soybean Glycine max (L.) Merr. breeding programs in Europe focused on development of elite non genetically modified (GM) cultivars for fast growing market of GM-free proteins for animal feed. Due to low variability of visual descriptors and mostly unknown pedigrees, divergent parents’ selection for crosses is a great challenge. Another challenge is cultivar distinction and protection of plant breeders’ rights of ever-increasing number of cultivars. By using 42 microsatellite (SSR) markers, we performed characterization of 97 commercial soybean cultivars and experimental lines developed at various research and breeding institutions in Europe (86) and in North and South America (11) in order to assess their genotype distinction power as well as utility for estimating genetic diversity and genetic structure. A set of 27 most polymorphic SSR markers was sufficient to discriminate all 97 genotypes. Discrimination of, by pedigree very related cultivars, was somewhat difficult due to the low polymorphism but still possible. Cluster analysis showed that European germplasm is mainly distributed into clusters reflecting breeding programs and maturity groups. Performed genetic characterization provides an insight into genetic structure of European soybean germplasm and might serve as a starting point for future breeding decisions. Disclosed SSR data of the analyzed commercial European germplasm can serve for genetic fingerprinting purpose as well as for foundation of public soybean cultivar database.