Feline immunodeficiency virus (FIV) was isolated from five Portuguese cats. The five strains were named RP1, PP2, TLP3, FP4 and CP5. The LTR, the CA region of the gag gene, and the V3-V5 region of the env gene were amplified by PCR, cloned and sequenced. The phylogenetic relationships among these three regions and other previously published sequences revealed an independent clustering of the Portuguese isolates in the LTR and CA protein. Based on the V3-V5 region the Portuguese isolates were classified as Subtype B, although they appear to be a subcluster within Subtype B. The study of these new FIV isolates showed the presence of Subtype B in Portugal and could provide a contribution for the understanding of FIV's genetic diversity.