Background: Cytochrome P450 2E1 (CYP2E1) is a main enzyme which plays a major role in activating and detoxifying many xenobiotics, carcinogens and drugs. Available studies suggest that CYP2E1 single nucleotide polymorphisms (SNPs) are involved in the risk of developing certain cancers after exposure to carcinogens. The purpose of the present study was to assess genotype and allele frequencies of polymorphic CYP2E1*5B in the Iranian population. Material and Methods: This study was performed on 200 healthy individuals (female: 100, male: 100) in medical laboratories of Ahvaz during 2011. The CYP2E1 *5B (rs3813867 G-1293C) assessment was carried out using PCR-RFLP method. The data were analyzed with ĸ2 and hardy-Weinberg Equation statistically methods. Results: The frequency of *1A/*1A (c1/c1), *1A/*5B (c1/c2) and *5B/*5B (c2/c2) genotypes was computed 97, 3 and 0 percent, respectively. The frequency of *1A (c1) and *5B (c2) alleles was computed 98.5 and 1.5 percent, respectively. No statistically significant difference was between two genders (p>0.05). Conclusion: The genotype distribution and allele frequencies of CYP2E1*5B polymorphism were similar to Turkish and some of the European populations. However, there are significant interethnic differences when the Iranian population is compared with the Eastern Asian, American and some of the European populations. The allelic distribution of this polymorphism did not vary with gender.