Feline leukemia virus (FeLV) is a retrovirus that affects domestic cats worldwide. It is a RNA virus but its genome is reverse transcribed into DNA that is integrated into the host's cell genome in the form of the proviral, where it remains probably life-long. FeLV infection may cause fatal illnesses, including neoplasia, bone marrow suppression, and immunodeficiency. Although there are very efficacious FeLV vaccines that led to a reduction in infection prevalence in many countries, FeLV still poses a problem to the infected individual but also to unprotected cat populations if the infection is not recognized promptly. This presentation focuses on the diagnosis of FeLV infection as well as on certain aspects of the pathogenesis that are essential for the comprehension of an optimal diagnostic workup. It provides current recommendations for diagnostic testing of FeLV infection and addresses potential diagnostic pitfalls and limitations of the different tests.