Current understanding of genetic variation in lions (Panthera leo) is inadequate to guide many management decisions necessary for conservation of the species. We studied sequence variation in the mitochondrial cytochrome-b (cyt-b) gene of 75 lions and nuclear variation at 11 microsatellite loci of 480 lions across 8 range states (Cameroon, Uganda, Kenya, Zambia, Zimbabwe, South Africa, Botswana, and Namibia) and 13 Lion Conservation Units (LCUs) plus two other unassigned sites (Cameroon and Zimbabwe). A total of 11 cyt-b haplotypes were found, whose variation follows an isolation-by-distance model. In combination with previously known sequences, the haplotypes document the close relationship, derived position, and limited variability of Asian and West and Central African lions relative to other extant lions. Both phylogenetic analyses and substitution networks identify two clades in Eastern and Southern Africa—one restricted to Namibia and South Africa and the other more widespread across the region. However, these analyses are equivocal on which of these is closest to the ancestor of modern lions. Microsatellite analyses showed high levels of variation within and among populations, subdivision among most LCUs, and evidence of isolation by distance. While rates of gene flow are generally low, admixture among lions in northern Botswana, Caprivi Strip (Namibia) and Zambia is apparent from STRUCTURE analyses. Conservation management plans should incorporate information on genetic variability and gene flow in delimiting management units and in guiding translocations of lions to minimize inbreeding and to control problem animals.