Chicken leukemia caused by avian leukemia virus has been troubling the global chicken industry, and its efficient and accurate detection and prevention can effectively reduce economic losses. This article aims to conduct pathogen analysis and identify a batch of suspected cases of chicken leukemia in a free-range chicken farm in Zhangshu City, Jiangxi Province, providing a basis for the disease diagnosis and treatment. Suspected chicken leukemia in broiler chickens was screened through clinical, pathological observations and H&E staining, then confirmed by PCR. According to clinical and anatomical diagnosis, the diseased chicken was weaker and unable to stand than the healthier broiler chicken of the same age. H&E staining showed heart, liver, spleen, lungs, and kidney lesions. The PCR and electrophoresis analysis detected a specific band of 405bp, which was consistent with the size of the target gene fragment. Therefore, these suspected cases were diagnosed as avian leukemia virus infection. The results indicated that the PCR is simple, direct, and highly accurate and can diagnose suspected cases of viral leukemia infection in farm chickens.