Abstract: In November 2020, 50-day-old chickens on a free-range chicken farm in Yichun City, Jiangxi Province suffered from indigestion, malaise, twitching before death, and "split posture" symptoms; some sick chickens had obvious tumors on their body surface. In order to diagnose the disease of affected chickens, autopsy lesions, histopathological observation and PCR detection methods were used for identification, and at the same time, the positive bands were sequenced and compared to construct an evolutionary tree. The results showed that a large number of gray white tumor foci could be seen in the heart, liver, kidney and lung of the dead chickens after autopsy, and the spleen was obviously enlarged. The pathological section of the submitted disease material conformed to the pathological characteristics of Marek’s disease. The pathogenic nucleic acid test showed that Marek’s disease virus, Avian leukemia virus, and avian Reticuloendotheliosis virus were positive. The evolutionary tree showed that the tested Marek’s disease virus was a virulent strain, but the comparison of the sequencing results of the positive bands showed that the sequence of avian Reticuloendotheliosis virus was highly similar to the Marek’s disease epidemic strain, which was a false positive result. The results suggested that the chickens of this chicken farm were infected with Marek’s disease virus and Avian leukemia virus. Scientific prevention and control could be adopted to improve the breeding environment, with a combination of vaccine immunization. Chicken farms should pay attention to the laboratory differential diagnosis, prevention and control of Marek’s disease.