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AIM: To investigate coxsackievirus B3 infection and its gene mutation in Keshan disease.METHODS: The expression of Coxsackievirus B3 RNA was detected in autopsy specimens of acute (12 cases), sub-acute(27 cases) and chronic (15 cases) Keshan disease by in situ hybridization. In sub-acute Keshan disease specimens, 3 cases with positive result by in situ hybridization were selected RT-PCR analysis. The DNA segments were then sequenced.RESULTS: Coxsackievirus B3 RNA was detected in the cytoplasm of myocardiocyte. The positive rate was 83% in acute, 67% in sub-acute and 80% in chronic Keshan disease.In the conservative region of Coxsackievirus B3 genome,there was a mutation in 234 (C-T) compared to the noncardiovirulent strain, CVB3/0.CONCLUSION: Coxsackievirus B3 RNA can survive and replicate in heart muscle of Keshan disease, which may play an important role in the occurrence of Keshan disease. The possible mechanism of occurrence of Keshan disease is associated with point a mutation in Coxsackievirus B3 genome.