To facilitate elucidation of the pathogenesis of alcohol dependence, we investigated the relationship between a genetic variant of diazepam biding inhibitor (DBI) C/A polymorphism (rs2276596) and alcohol dependence. We determined the DBI genotypes using a novel method involving PCR-RFLP in healthy controls and alcoholics with a diagnosis of alcohol dependence by ICD-10 (F10.20). There was a significant difference in the rs2276596 polymorphism C/A allele frequency of the DBI gene (P < 0.0001) between alcoholics and healthy controls. The present data suggested that a mutant allele of the DBI was one of the risk factors for alcohol dependence as for the rs2276596 polymorphism.