Spillover from domestic species has been frequently implicated as a source of disease in wildlife populations. Previous study of wild ocelots (Leopardus pardalis) has demonstrated that populations in close proximity to human settlements can be exposed to multiple pathogens of domestic dogs and cats. In contrast, prior serosurveys have determined that ocelots have an extremely low level of feline immunodeficiency virus (FIV)-like infection (0–5%). This low seroprevalence is remarkable because other wild felids have species-specific FIV infection rates approaching 100%. This study was designed to determine the seroprevalence of domestic animal pathogens and feline lentivirus (FIV) in an isolated island population of ocelots that has practically no contact with domesticated species. We collected serum samples from 12 ocelots native to Barro Colorado Island (BCI) and screened for antibodies to: (1) feline calicivirus, canine distemper virus, and feline herpesvirus using serum neutralization; (2) feline coronavirus using immunofluorescence assay; (3) feline parvovirus using hemagglutination inhibition; and (4) feline lentivirus using immunoblots with virus from three antigenically distinct FIV strains (puma lentivirus, lion lentivirus, and feline immunodeficiency virus). Sera from naïve or exposed animals were used as controls. All 12 individuals were seronegative for exposure to all pathogens with the exception of lentivirus infection. Five of seven females were positive for lentiviral infection, and one female’s serum was inconclusive. Interestingly, only one of five males was positive for lentiviral-reactive antibodies. These results are suggestive of female ocelot predilection for lentiviral infection but were only marginally significant when analyzed using non-parametric methods (p=0.08). Gender bias for lentiviral infection has not been reported previously in FIV infection, and should such a relationship be validated by analysis of additional samples, this population would provide a rare opportunity to determine behavioral or ecological factors underlying this observation. Overall, these results suggest that isolation from domestic reservoirs will prevent the introduction of disease in small, high-density populations of wild felids. We speculate that lentiviral seropositivity in this population represents infection with a species-specific virus that was enzootic prior to isolation of the BCI ocelot population. We hypothesize that the high density of ocelots on BCI leads to greater contact rates among individuals and may explain the higher seroprevalence of lentivirus exposure in this population as compared to previously studied populations.