The characterisation of two distinct feline foamy virus sequence groupings in the external surface portion of the viral env gene are reported. Although amino acid identities in the Gag nucleocapsid, Pol protease, and Env transmembrane domains were greater than 92% in the 12 proviral sequences examined, two distinct sequence groups were observed in the Env surface (SU) protein. Only 57% amino acid identity was observed in the Env SU between the two groups designated FUV7-like or 951-like, while within these groups > 97% identity was found. Isolates FUV7 and 951 represent two serogroups previously characterised by Flower et al. (1985). A 100% correlation was found among FeFV seroreactivity, virus isolation, and detection of viral DNA in feline leucocytes using a single round of PCR amplification. Serum neutralisation assay using autologous virus, as well as isolates 951 and FUV7, revealed that viruses with FUV7-like sequences were in a single neutralisation group and viruses with 951-like sequences were in a single neutralisation group. Based on these results, group-specific PCRs were developed, using the same sense primer with an antisense primer specific for each group. Using he PCR, no evidence of superinfection of any cat with virus from both serogroups was detected.