Feline immunodeficiency virus is a retrovirus of domestic cats that causes immunosuppressive disease and lifelong infection. Lentivirus has also been detected in African lions (Panthera leo). The lentivirus infecting lions in southern Africa has never been isolated; thus, knowledge about its molecular characteristics in these populations is limited. Our investigation used whole blood samples collected opportunistically from free-ranging southern African lions in Kruger National Park, South Africa, and from Hlane Royal National Park in Swaziland to analyse the lentivirus. Whole blood samples from captive exotic felids from zoos in South Africa and the United States, and from domestic cats in South Africa were analysed for comparison. A nested polymerase chain reaction (PCR) assay was used to amplify a portion of the proviral DNA encoding the reverse transcriptase. The nucleotide sequence of all products was determined and examined. The PCR assay was successful in amplifying lion lentivirus, with 52 positive and 65 negative samples. Of the 34 sequences amplified from a variety of felids, six showed an average of 96% homology to domestic feline lentivirus, and 28 showed an average of 94% homology to lion lentivirus. In addition, domestic cat lentivirus nucleic acid was amplified from a captive tiger, demonstrating the possibility of cross-species transmission.