Abstract <p>The impact of genetic mechanisms should be clearly understood before clinical practice of drugs since they could cause variations in biotransformation, clearance, and thus, the optimal dose of drugs. Naltrexone, an opioid antagonist metabolized by AKR isoenzymes, is used to manage opioid and alcohol use disorders. Although its high efficacy, adherence to treatment with naltrexone was different between patients due to most likely patients’ genetic information. The aim of this study is to develop novel PCR-RFLP assays to genotype <i>AKR1C3</i> rs3763676 and rs12529 polymorphisms and to find out the allele and genotype frequencies of these polymorphisms in a Turkish population. Unrelated volunteers (<i>n</i> = 222) between 18–65 years were analyzed to determine genotype and allele frequencies of <i>AKR1C3</i> rs3763676 and rs12529 in a Turkish population with novel PCR-RFLP assays. The minor allele frequencies (MAF) of <i>AKR1C3</i> rs3763676 and rs12529 polymorphisms were 24.0% and 56.0%, respectively. MAF of <i>AKR1C3</i> rs12529 in Turkish population was higher than those of in African and in Taiwanese, respectively. The frequency of T (variant) allele of <i>AKR1C3</i> rs3763676 was higher in Korean as compared to Turkish. <i>AKR1C3</i> rs3763676 and rs12529 allele variants were not in linkage disequilibrium (<i>D</i> = 0.57, <i>R</i>² = 0.08). PCR-RFLP assays developed in the study and the documented allele frequencies will be useful for direct detection of <i>AKR1C3</i> rs3763676 and rs12529 variations in pharmacogenetic studies of naltrexone pharmacology.</p>