Feline immunodeficiency virus (FIV) is responsible for immunodeficiency syndrome in cats. Several viral subtypes have been identified, each with a variable geographical distribution. To date, the subtype B is known to be the genotype spread in Italy. In this study, the genetic diversity of FIV in northern Italy was assessed by detecting proviral DNA in the blood samples of 50 cats determined to be positive through an anti-FIV antibodies test. These cats were tested using six different PCR assays, and the identified viruses were sequenced and analyzed. Forty-eight cats were confirmed positive, and several FIV subtypes were characterized. As expected, the subtype B was the most commonly observed, and the subtype A was reported for the first time in Italy. Moreover, a new taxon possibly representing an additional FIV subtype was detected, and one virus belonging to subtype B potentially had a recombinant origin. The genetic variability between the FIV viruses that emerged in this study may lead to the potential diagnostic failure of single molecular tests. Therefore, a new diagnostic strategy, which adopts different molecular tests and sequencing, is recommended to monitor the evolution and spread of FIV.