Background: According to World Health Organization (WHO) report about 400 million people are chronically infected with hepatitis B virus (HBV). Host immune responses which are mainly controlled by cytokines, can be either effective in disease progression or control the infection. Interleukin-27 (IL-27) is a pro-inflammatory cytokine which promotes Th1 responses. Genetic variations (e.g. single nucleotide polymorphisms [SNPs]) can affect the product or activity of IL-27 gene. The aim of present study was to determine the association between IL-27 rs153109 and chronic HBV infection among the Iranian population. Materials and Methods: In this study chronic HBV patients (n=120, Anti-HBc Ab positive and HBsAg positive for more than 6 months) and controls (n=120) from healthy individuals referred to Tehran Taleghani hospital (2013-2014) were studied. Genotypes of IL-27 gene polymorphism were detected by PCR-RFLP. DNA sequencing was applied on 10% of samples to validate the genotyping results. The studied variables were polymorphism genotypes/alleles, clinical status, age and gender. Results: Results showed no statistically significant difference for patients and control groups neither in genotype frequencies of AA among the chronic group (30%) compared to healthy controls (32.5%) (P=0.368); nor in allele frequency A) 60.4%) for patients against A 59.2% in control groups (P=0.780). Conclusion: Despite the importance of IL-27 in the immune response, the findings of this study suggests that genetic variants of IL-27 SNP 153109A/G were not associated with susceptibility to the chronic infection of HBV.