The role of genetic relatedness in the evolution of eusociality has been the topic of much debate, especially when contrasting eusocial insects with vertebrates displaying reproductive altruism. The naked mole-rat, Heterocephalus glaber, was the first described eusocial mammal. Although this discovery was based on an ecological constraints model of eusocial evolution, early genetic studies reported high levels of relatedness in naked mole-rats, providing a compelling argument that low dispersal rates and consanguineous mating (inbreeding as a mating system) are the driving forces for the evolution of this eusocial species. One caveat to accepting this long-held view is that the original genetic studies were based on limited sampling from the species’ geographic distribution. A growing body of evidence supports a contrary view, with the original samples not representative of the species—rather reflecting a single founder event, establishing a small population south of the Athi River. Our study is the first to address these competing hypotheses by examining patterns of molecular variation in colonies sampled from north and south of the Athi and Tana rivers, which based on our results, serve to isolate genetically distinct populations of naked mole-rats. Although colonies south of the Athi River share a single mtDNA haplotype and are fixed at most microsatellite loci, populations north of the Athi River are considerably more variable. Our findings support the position that the low variation observed in naked mole-rat populations south of the Athi River reflects a founder event, rather than a consequence of this species’ unusual mating system.