The black rhino sanctuaries system has played a key role in repopulating and starting new subpopulations in Kenya. If this system is efficiently managed it may save the black rhinoceros from local extinction. Understanding the genetic status of endangered species is the most elemental sine qua non of animal breeding and conservation. It is therefore important to determine the genetic diversity of black rhino populations, especially of nucleus breeding populations that are used as a source of individuals for translocation and supplementation programs. We assessed the genetic diversity of one of the pioneer breeding subpopulations of the black rhino Diceros bicornis in Kenya using a mitochondrial DNA D-loop region. We then compared this subpopulation with the entire Kenyan population to determine its status vis-à-vis the Kenya pooled population and determine the possible sources/relationships of the founder individuals. In the 469-bp D-loop region we analyzed, 7.11% of the sites were variants, contributing to 18 distinct haplotypes. Estimates of genetic diversity using haplotype and nucleotide diversity metrics showed that the Lake Nakuru National Park subpopulation has a slightly lower genetic diversity when compared with that of the pooled Kenya population. The phylogenetic tree revealed that Lake Nakuru National Park founder individuals were probably sourced from multiple subpopulations. The dendrogram and the principal coordinate analysis plot indicated that the Maasai Mara subpopulation is not a distinct subpopulation, as had been suggested previously. Our results provide baseline genetic data for the Lake Nakuru National Park breeding subpopulation and valuable information for translocation/supplementation programs.