This study aimed at characterising the genetic diversity of two Fipa cattle populations (Sumbawanga and Nkasi) of South-Western Tanzania, and establishing their genetic relationships with the other indigenous cattle strains (Tarime, Iringa red and Ankole) and Friesian cattle found in the area. The genetic diversity was analysed using 30 microsatellite markers. All the markers used were highly polymorphic. The Nkasi Fipa cattle exhibited the highest mean number of alleles (7.31) and mean genetic diversity (0.732) per locus, followed by Sumbawanga Fipa cattle with 7.10 mean number of alleles and 0.725 mean genetic diversity per locus, with the latter population having a very low mean inbreeding coefficient (FIS = 0.027). Three percent of the genetic diversity was due to differences among indigenous strains while the rest was due to differences among individuals within the strains. Small genetic distances (DA) were observed between Sumbawanga Fipa and Nkasi Fipa (0.032), Tarime (0.073), Iringa red (0.076) and Ankole cattle (0.086). As expected, the largest genetic distances were observed between the Friesian and all indigenous strains since this breed has a quite distinct genetic origin. In the assignment test, the proportion of animals from each group correctly assigned to their source population ranged from 55.3 percent (for Nkasi Fipa) to 100 percent (for Friesian). Despite the low genetic differentiation and genetic indistinctiveness of the Sumbawanga Fipa population from the other indigenous strains, its high genetic diversity, very low inbreeding coefficient and a threat emanating from population admixture with other indigenous strains underscore the importance of establishing appropriate conservation and management strategies for it.