The decline of lions (Panthera leo) in Kenya has raised conservation concerns about their overall population health and long-term survival. This study aimed to assess the genetic structure, differentiation and diversity of lion populations in the country, while considering the influence of past management practices. Using a lion-specific Single Nucleotide Polymorphism (SNP) panel, we genotyped 171 individuals from 12 populations representative of areas with permanent lion presence. Our results revealed a distinct genetic pattern with pronounced population structure, confirmed a north-south split and found no indication of inbreeding in any of the tested populations. Differentiation seems to be primarily driven by geographical barriers, human presence and climatic factors, but management practices may have also affected the observed patterns. Notably, the Tsavo population displayed evidence of admixture, perhaps attributable to its geographic location as a suture zone, vast size or past translocations, while the fenced populations of Lake Nakuru National Park and Solio Ranch exhibited reduced genetic diversity due to restricted natural dispersal. The Amboseli population had a high number of monomorphic loci likely reflecting a historical population decline. This illustrates that patterns of genetic diversity should be seen in the context of population histories and that future management decisions should take these insights into account. To address the conservation implications of our findings, we recommend prioritizing the maintenance of suitable habitats to facilitate population connectivity. Initiation of genetic restoration efforts and separately managing populations with unique evolutionary histories is crucial for preserving genetic diversity and promoting long-term population viability.