Urochloa (syn.-Brachiaria s.s.) is one of the most important tropical forages that transformed livestock industries in Australia and South America. Farmers in Africa are increasingly interested in growing Urochloa to support the burgeoning livestock business, but the lack of cultivars adapted to African environments has been a major challenge. Therefore, this study examines genetic diversity of Tanzanian Urochloa accessions to provide essential information for establishing a Urochloa breeding program in Africa. A total of 36 historical Urochloa accessions initially collected from Tanzania in 1985 were analyzed for genetic variation using 24 SSR markers along with six South American commercial cultivars. These markers detected 407 alleles in the 36 Tanzania accessions and 6 commercial cultivars. Markers were highly informative with an average polymorphic information content of 0.79. The analysis of molecular variance revealed high genetic variation within individual accessions in a species (92%), fixation index of 0.05 and gene flow estimate of 4.77 showed a low genetic differentiation and a high level of gene flow among populations. An unweighted neighbor-joining tree grouped the 36 accessions and six commercial cultivars into three main clusters. The clustering of test accessions did not follow geographical origin. Similarly, population structure analysis grouped the 42 tested genotypes into three major gene pools. The results showed the Urochloa brizantha (A. Rich.) Stapf population has the highest genetic diversity (I = 0.94) with high utility in the Urochloa breeding and conservation program. As the Urochloa accessions analyzed in this study represented only 3 of 31 regions of Tanzania, further collection and characterization of materials from wider geographical areas are necessary to comprehend the whole Urochloa diversity in Tanzania.