Feline immunodeficiency virus (FIV) is a lentivirus of domestic cats worldwide. Diagnosis usually relies on antibody screening by point-of-care tests (POCT), e.g., by enzyme-linked immunosorbent assays (ELISA), and confirmation using Western blot (WB). We increasingly observed ELISA-negative, WB-positive samples and aimed to substantiate these observations using 1194 serum/plasma samples collected from 1998 to 2019 primarily from FIV-suspect cats. While 441 samples tested positive and 375 tested negative by ELISA and WB, 81 samples had discordant results: 70 were false ELISA-negative (WB-positive) and 11 were false ELISA-positive (WB-negative); 297 ambiguous results were not analyzed further. The diagnostic sensitivity and specificity of the ELISA (82% and 91%, respectively) were lower than those reported in 1995 (98% and 97%, respectively). The diagnostic efficiency was reduced from 97% to 86%. False ELISA-negative samples originated mainly (54%) from Switzerland (1995: 0%). Sixty-four false ELISA-negative samples were available for POCT (SNAP /WITNESS ): five were POCT-positive. FIV RT-PCR was positive for two of these samples and was weakly positive for two ELISA- and POCT-negative samples. Low viral loads prohibited sequencing. Our results suggest that FIV diagnosis has become more challenging, probably due to increasing travel by cats and the introduction of new FIV isolates not recognized by screening assays.