ABSTRACT Avian leukosis virus (ALV) subgroups A, B, and J are very common in poultry flocks and have caused serious economic losses in recent years. A multiplex PCR (mPCR) method for the detection of these three subgroups was developed and optimized in this study. We first designed a common forward primer, PF, and three downstream primers, AR, BR, and JR, which can amplify 715 bp for subgroup A, 515 bp for subgroup B, and 422 bp for subgroup J simultaneously in one reaction. The mPCR method produced neither cross-reactions with other subgroups of ALVs nor nonspecific reactions with other common avian viruses. The detection limit of the mPCR was as low as 1 × 10 ³ viral DNA copies of each of the three subgroups. In animal experiments, the mPCR detected ALVs 2 to 4 days earlier than did virus isolation from whole-blood samples and cloaca swabs. Furthermore, a total of 346 clinical samples (including 127 tissue samples, 86 cloaca swabs, 59 albumen samples, and 74 whole-blood samples) from poultry flocks with suspected ALV infection were examined by mPCR, routine PCR, and virus isolation. The positive sample/total sample ratios for ALV-A, ALV-B, and ALV-J were 48% (166/346) as detected by mPCR and 48% (166/346) as detected by routine PCR. However, the positive sample/total sample ratio detected by virus isolation was 40% (138/346). The results of the mPCR and routine PCR were confirmed by sequencing the specific fragments. These results indicate that the mPCR method is rapid, specific, sensitive, and convenient for use in epidemiological studies of ALV, clinical detection of ALV, and ALV eradication programs.