Opioid Use Disorder (OUD) has been linked to dopamine and the neurological reward centers. Methylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in the production of many neurotransmitters such as dopamine. As such, MTHFR variants that lead to decreased production of neurotransmitters may play a role in OUD. However, lacunae exist for characterizing the prevalence of the MTHFR mutations in an OUD population. The objective of this study was to determine prevalence of the MTHFR gene mutations in a rural Tennessean population with OUD. This study was a retrospective cohort of individuals with OUD that evaluated the prevalence of MTHFR variants. Patients were categorized as normal, homozygous C677T, heterozygous C677T, homozygous A1298C, or heterozygous A1298C. The primary outcome was a qualitative comparison of the prevalence of each of the MTHFR variants in our cohort to the publicly reported MTHR polymorphism prevalence. Secondary outcomes include race and ethnicity differences as well as stimulant use differences for each of the variants. A total of 232 patients undergoing care for opioid use disorder were included in the study. Of those included, 30 patients had a normal MTHFR allele and 202 had a variant MTHFR allele. Overall, the prevalence of any MTHFR variant was 87.1% (95% CI 82.6-91.4%). When comparing those with a normal MTHFR allele to those with any MTHFR variant, there was no difference in age, sex, race and ethnicity, or stimulant use. The overall prevalence of MTHFR variants in patients with opioid use disorders is high.