Background/Objectives: Methadone maintenance treatment (MMT) is widely used in opioid use disorder (OUD). Its efficacy is influenced by its metabolism, primarily mediated by Cytochrome P450 (CYP450) enzymes in the liver. Genetic polymorphisms in CYP450 genes and other factors, such as age, sex, and concomitant treatments, contribute to interindividual variability in methadone response. This article addresses the relevance of pharmacokinetic biomarkers in methadone metabolism and its impact on treatment outcomes in European populations over the past 25 years.

Methods: A systematic review was conducted using four databases (PsycINFO, PubMed, Scopus, and Web of Science) for studies published between 2000 and 2024 following the PRISMA 2020 guidelines (CRD42025641373 in PROSPERO). Two independent reviewers screened and assessed the study quality using NHLBI tools. Discrepancies were solved through consensus. Relevant data including sample size, genetic biomarkers, and key findings were extracted for each study. Data were synthesized and described in detail.

Results: Fourteen studies on pharmacogenetic biomarkers influencing methadone metabolism in European populations were analyzed, encompassing a total of 3180 subjects. *6 was identified as a key variant associated with increased (S)-methadone plasma levels, potentially leading to cardiac complications, while the role of other pharmacokinetic genes, including and , was inconclusive.

Conclusions: Genetic polymorphisms significantly influence methadone metabolism, with the *6 allele playing a key role in (S)-methadone metabolism and associated with cardiac risks. Pharmacogenetic studies integrating co-mediation-the principal cause of phenoconversion-as a potential variable alongside gender differences and encompassing adequate sample sizes could improve outcomes and establish the basis for personalized medicine of MMT.