Introduction: The CYP2D6 gene, a key enzyme in drug metabolism, exhibits high polymorphism. Variants rs1065852 (CYP2D6*10) and rs1135840 (CYP2D6*41) are associated with reduced enzyme activity, making their precise detection crucial for optimizing pharmacotherapy. This study aimed to develop and validate plasmid-derived reference materials for detecting these Single-Nucleotide Polymorphisms (SNPs) via real-time PCR genotyping. Methods: Recombinant plasmids carrying wild-type and mutant-type sequences for rs1065852 (100C > T) and rs1135840 (4180G > C) were constructed in E. coli DH10B. Target sequences were verified by PCR and Sanger sequencing. Analytical performance was evaluated for linearity, Limit of detection (LoD), allelic discrimination, homogeneity, genetic stability (15 generations), and storage stability (up to 180 days). Cross-platform compatibility was assessed using different SNP genotyping assays and real-time PCR instruments. Results: PCR and sequencing confirmed correct SNP integration. qPCR demonstrated strong linearity (R2 ≥ 0.9874) with an LoD of 103 copies/reaction. Allelic discrimination was distinct and reproducible. Homogeneity testing yielded coefficients of variation below 5%. The plasmids-maintained stability across generations and storage conditions, with consistent SNP calls across platforms and assays. Discussion: The developed plasmid-based reference materials exhibited high analytical perfor-mance, stability, and reproducibility. Their cross-platform compatibility enhances their utility in standardizing CYP2D6 SNP detection, addressing variability in pharmacogenomic assays. Conclusion: Validated plasmid-based reference materials for rs1065852 and rs1135840 offer a cost-effective, accurate, and stable standard for pharmacogenomic testing, supporting improved assay precision and broader clinical implementation. 2026, Bentham Science Publishers