To investigate the co-infection status of avian leukaemia viruses (ALV) in layer flocks in China, 184 samples were collected from suspected ALV-J infection cases in Ningxia, Hubei, Guangdong, Shandong, Liaoning, Jilin and Heilongjiang provinces. These samples were subjected to PCR, virus isolation and indirect immunofluorescence assay (IFA) to detect infection by subgroup J ALV (ALV-J) in layer chicken flocks and co-infection with Reticuloendotheliosis (REV), Marek's disease virus (MDV) or Chicken anemia virus (CAV). The results showed that infection of ALV-J exists in all seven provinces with 60.9 % samples detected positive in 82.1 % flocks. The co-infection with REV, MDV or CAV with ALV-J accounted for 13.6 %, 24.5 %, 22.8 %, respectively. Multiple infection by up to 4 different viruses was detected in some cases. These results indicated there was serious ALV-J infecton in layer flocks in China and co-infection with other immunosuppressive virus is prevalent, therefore it is strongly recommended more attention be paid on co-infection in clinical diagnosis of ALV-J infection in layer flocks in the future.