Enzyme-linked immunosorbent assay (ELISA) has high sensitivity and specificity for detection of feline immunodeficiency virus (FIV) antibodies, but does not distinguish between infection and vaccination. Real-time quantitative PCR (qPCR) assays may distinguish infected from vaccinated cats. Performance of a commercial qPCR assay was assessed with blood samples from 29 FIV-infected non-vaccinated, 26 FIV-uninfected vaccinated and 35 FIV-uninfected non-vaccinated cats. FIV infection status of cats was assigned based on a combination of vaccination and medical history, prevention of contact with potentially infected cats and two FIV antibody ELISA results. Test sensitivity and specificity were determined against this gold standard. The qPCR test yielded positive results in samples from 23/29 FIV-infected non-vaccinated, 2/26 FIV-uninfected vaccinated and 0/35 FIV-uninfected non-vaccinated cats. It was concluded that the qPCR test was moderately sensitive and highly specific for the diagnosis of FIV infection, and that it may be suitable for ruling out FIV infection in cats with a positive antibody ELISA result and unknown vaccination history.