Background- The African lion (Panthera leo) is listed as vulnerable by the IUCN Red List, mainly threatened by prey base depletion and indiscriminate killing, as a result of retaliatory or pre-emptive killing to protect human life and livestock. Additionally, habitat loss by land degradation and conversion has led to the isolation of subpopulations, potentially decreasing gene flow resulting from weakened connectivity and leading to genetic drift. In the present study, we investigated the evolutionary history and structure of the species at different spatiotemporal scales. Results- The mitochondrial cytochrome b gene (N = 128), 11 microsatellites (N = 103) and 9,103 SNPs (N = 66) were investigated, including a large sampling from Tanzania, since it hosts the largest lion population among all African lion range countries. In addition to supporting the dichotomic continental scale structure (i.e. West-Central vs East-Southern, which is also found in many other savanna mammals with large distribution ranges), three lion clusters could be identified in Tanzania. The clusters are geographically distributed in the northern, southern and western regions of the country. The genetic differentiation between these clusters appears to have resulted from the combined effects of recent anthropogenic pressure and environmental/climatic factors. Significance- The Tanzanian annual human population growth of 3.1% indicates that the human pressure on wildlife habitats is not expected to decrease in the short-term, resulting in an increased human encroachment on landscapes. Since male lions have relatively weak dispersal capabilities, and since the species barely co-exist with humans, further fragmentation of its habitat could lead to an even greater loss of connectivity between the mosaic of protected areas, which might lead to a rapid loss of genetic variability. Continuous monitoring of the identified strongholds would be highly recommended, especially since all three clusters have undergone recent demographic contraction, as supported by the census records.