Background: Methadone therapy is a major protocol in opioid addiction cases in many health care systems. Population-based studies have shown that in addicted people, the genetic profile affects their response to methadone therapy. Therefore, this study designed to examine the frequency of two SNPs of the CYP2B6 gene (rs3745274 and rs3211371) in addicted cases in two methadone-responders and methadone non-responders groups. Methods: A total of 199 opioid-addicted individuals and 117 unaffected control subjects were genotyped for rs3745274 and rs3211371 polymorphisms of the CYP2B6 gene using the tetra-primer amplification refractory mutation system-polymerase chain reaction. Results: Results of this study revealed the significant association of rs3745274 GG (p < 0.001; OR = 0.027; 95% CI = 0.14-0.49) and GT (p < 0.001; OR = 4.04; 95% CI = 2.26-7.21) genotypes with the risk of addiction in methadone-responders. Also, a significant association between rs3745274 GG (p < 0.001; OR = 0.28; 95% CI = 0.15-0.51) and GT (p < 0.001; OR = 5.1; 95% CI = 2.8-5.28) genotypes and addiction relapse was found in methadone non-responders. Conclusion: Based on our findings, we can conclude that rs3745274 variant of CYP2B6 gene could serve as a potential biomarker, to evaluate the prognosis of addicted people fate under treatment with methadone.