The African lion (Panthera leo) is listed as vulnerable by the IUCN Red List. The species is mainly threatened by indiscriminate killing, primarily as a result of retaliatory or pre-emptive killing to protect human life and livestock, and by prey base depletion. In addition, habitat loss and its conversion into farmland have led to a number of subpopulations becoming small and isolated. As a consequence of the weakened connectivity between the main strongholds, genetic drift and loss of genetic diversity could affect the long-term survival of the species. In the present study, we investigated the evolutionary history of P. leo at different temporal and spatial scales. A total of 182 individuals were used, 77 of which came from Tanzanian protected areas. The mitochondrial cytochrome b gene was sequenced and the specimens were genotyped for 11 microsatellites and more than 9,000 SNPs through the Genotyping by Sequencing methodology. Results indicate that the African lion is structured into two lineages at the continental scale (West-Central vs South-Eastern), a pattern observed within many other large savanna species displaying large distribution ranges. Pleistocene climatic oscillations and biogeographical barriers were proposed as the main factors to have driven the lineage sorting. The SNPs in addition, allowed for the identification of 3 lion populations in Tanzania (STRUCTURE), geographically structured between the North, South and West-Southern regions. Overall, the Tanzanian populations displayed good levels of genetic diversity with limited signs of inbreeding, while their effective population sizes were shown to have been decreasing over the last 70 generations. Since the uncovered population structure did not seem to emerge from isolation-by-distance (IBD, spatial autocorrelation) and was shown to be of recent origin (DIYABC), recent land conversion and agricultural activities appear to best explain the genetic population differentiation within the African lion of Tanzania. Using various molecular markers, the present work will further explore the evolutionary history of the species to bring new insights in its conservation requirements.