Objective：To establish a method to detect mutation sites of hepatitis B virus（HBV） preS gene by reverse dot blot（RDB）.Methods：Oligonucleotide probes were designed according to the codon mutations at the 35th，39th，48th，155th and 165th sites，re－spectively. Mutated sites from 38 patients with HBV were detected by RDB and were compared with the sequencing results. Results：Coincidence between RDB detection results and DNA sequencing was 100%. Conclusions：Detection of the preS gene mutation by RDB is a cost-effective，rapid and reliable method，which is of significance in clinical testing for HBV preS mutations and determin－ing the efficacy of HBV infection and prognosis of patients.