To obtain more insights into the prevalence and diversity of species-specific Feline Immunodeficiency Virus (FIV) strains in naturally occurring felid species, 26 leopards (Panthera pardus) from the Kruger National Park (KNP), South Africa, were sampled. Prevalence was determined using a PCR protocol designed to target a 577 bp fragment in the pol-RT gene. Overall prevalence of FIV_Ppa was estimated at 73%, with no difference in prevalence between male and female leopards. Consistent with previous FIV studies on other felid species, prevalence appears to increase with age (adult = 84%; subadult = 43%). Phylogenetic analyses of these novel sequences were conducted against a revised FIV pol-RT species-specific reference dataset using both Bayesian and maximum likelihood methods. Within FIV_Ppa two distinct evolutionary groupings are present, which suggests the possibility of geographic variation within FIV_Ppa and the possibility of distinct subtypes, similar to what has been found in lions (Panthera leo) and domestic cats (Felis catus). The larger FIV_Ppa dataset provides newinsights into the epidemiology of this under-studied FIV strain and with such high prevalence rates, further studies should focus on immunological and clinical consequences of FIV in wild felids.