Pharmacogenomics enables the prediction of individual drug responses based on genetic profiles, representing a critical step toward personalized medicine through routine genetic testing. Advances in high-throughput sequencing technologies have facilitated the simultaneous identification and interpretation of variants across multiple pharmacogenes. However, incorporating pharmacogenomics into clinical practice remains challenging, partly due to limited knowledge of population-specific pharmacogenetic data. This study aimed to construct the most comprehensive pharmacogenomic map of the Serbian population to date. Genomic data from 881 Serbian individuals, obtained via clinical and whole exome sequencing, were analyzed. Raw sequencing data were processed using a custom pipeline for alignment and variant calling. Pharmacogenetic star alleles and predicted phenotypes were annotated using PharmCAT and Stargazer. Frequencies of star alleles and phenotypes were calculated and compared to both European and global populations. Population differentiation was assessed through Wright’s fixation index (F_ST). The analysis revealed the greatest population differentiation between Serbians and the global population. Among Serbians, key pharmacogenes with notable star allele frequencies and clinically actionable phenotypes included CYP2B6, NAT2, SLCO1B1, UGT1A1, and VKORC1, exhibiting distribution patterns significantly distinct from other European populations. The observed variations in pharmacogenetic phenotypes, which impact responses to drug classes such as statins and antidepressants, highlight the potential benefit of integrating drug-response data into genetic reports for the Serbian population. Pharmacogenomic implementation could be efficiently achieved using existing clinical and whole exome sequencing datasets.
