Abstract: In order to investigate the prevalence status of avian leukosis virus infection in Shandong province, 3 882 serum samples, 2 428 cloacal swab samples and 41 suspectedcase samples were collected from different areas of Shandong province for respectively detection through methods of serology, pathology and etiology. The test results indicated that P27 antigen and ALV antibody were positive in all stains of layer chickens including grandparent flock, the p27 antigen and antibody’s positive rates of ALVA/B, ALVJ, REV were 19.36%, 9.29%, 5.18%, 1377%, respectively. The main groups getting disease were Commercial flock and parent flock, while there’re also some Hyline brown grandparent flock got disease. The pathology diagnosis indicates that the tumors mainly include myelocytoma (27/41), hemangioma (7/41), fibrosarcoma (2/41), leiomyosarcoma (2/41) and MD (5/41). Among the 41 samples, 33 samples were positive for ALVJ (80.49 %), and 22 samples were positive for MDV (53.66%), the coinfection rate was 43.9%. Among 17 virus stains isolated, the sequence homology of gp85 gene of ALVJ were ranged from 94.0% to 100%. The 17 isolated virus stains shared 94.3% to 98.7% identity with HPRS103, and shared low homology with other strains (84.4%96.8%). These results indicated that ALV infection existed in all stains of layer chickens in Shandong province and coinfection with MDV, REV were occured. Myelocytoma and hemangioma were the main clinical symptoms of sick chickens.