Objective To investigate the feasibility of reverse transcription_polymerase chain reaction (RT_PCR) to evaluate the inactivation efficacy of viruses in water, and to discuss the mechanisms of HAV inactivation by chlorine. Methods Cell cultrue, ELISA method and long_overlapping RT_PCR were developed to detect the infectivity, antigenicity and entire genome of HAV inactivated by chlorine. Results The cell culture results revealed that the infectivity was completely inactivated after exposure to 10 mg/L or 20 mg/L of cholrine for more than 30 minutes, the antigenicity was completely inactivated after exposure to 10 mg/L of chlorine for 60 minutes. The 5' nontranslated region (5'NTR) of neculear acids of HAV was the most sensitive to chlorine, which was confirmed with the inactivation of infectivity of HAV. Conclusions The results implied that the inactivation of HAV by chlorine was due to the loss the 5' NTR. It was believed that PCR could be used to assess the efficacy of disinfection of HAV by chlorine and also could be applied to research the inactivation mechanisms of viruses by disinfectants.