Indigenous livestock resources are strategic in the socio-economics of rural agricultural systems to ensure food security in resource-poor countries. Therefore, better understanding of genetic variation holds the key to future utilization through conservation. We report the first analysis of genetic diversity of Nigerian sheep based on the D-loop region of the Ovis aries mitochondrial genome using 1 179 bases between sites 15 437 and 16 616 base pairs. A sample of 290 animals made up of Balami, West African Dwarf (WAD), Uda and Yankasa breeds were randomly collected from across Nigeria. Ninety-six haplotypes were observed with a high mean haplotype diversity of 0.899 ± 0.148. Gene diversity was highest in Uda (0.921 ± 0.021) and lowest in WAD (0.852 ± 0.061). Population specific FST indices varied from 0.00133 in Uda to 0.00335 in WAD. Yankasa had the highest number of polymorphic sites (201), while the least was in Uda (96). Analysis of molecular variance revealed that 0.23 percent of the variation is found among populations compared with 99.77 percent variation found within populations. The phylogenetic tree indicates that the mitochondrial lineages of these sheep breeds originated from a common source consistent with first divergence of Yankasa followed by WAD, while Balami and Uda remain more closely related. These results suggest that evolutionary divergence of Nigerian sheep breeds based on mitochondrial DNA D-loop sequence may be coincident with geographical distribution in Nigeria and suggest significant interbreeding. This could have implications for managing improvement and conservation strategies and long-term conservation of Nigerian indigenous sheep.