Genetic polymorphism can cause variation in tramadol (TR) pharmacokinetic characteristics and the expected clinical response. In forensic toxicology, the data about parent and metabolite concentrations (MRs
metabolic ratios) could facilitate to determine the cause of death and to assess time between drug intake and death. In this study, the aim was to investigate if UGT1A8, UGT2B7, ABCC2, and SLC22A1 genotyping can facilitate interpretation by investigating the frequency of UGT1A8, UGT2B7, ABCC2, and SLC22A1 genotypes in forensic autopsy cases positive for TR and to assess whether there is a correlation between these genetic variants and MRs. Cases positive for TR (n = 48) were genotyped by HaloPlex Target Enrichment system for UGT1A8, UGT2B7, ABCC2, and SLC22A1 sequencing, in order to identify single nucleotide polymorphisms (SNPs) and/or insertion deletion (INDELs). In addition to, the concentrations of TR and its metabolites (M1 & M2) were determined by LC-MS/MS. Cases were categorized by cause of death. The investigated SNPs/INDELs were not overrepresented in any group. We found significant correlations between several loci (12 out of 73) in UGT1A8, ABCC2, and SLC22A1 genes and MRs (M2/M1, TR/M2, and TR/M1) in post-mortem TR cases. These results indicate these polymorphisms in the 4 investigated genes might influence TR pharmacokinetics leading to an unsatisfactory therapeutic effect or increasing the risk of toxicity. However, these findings should be supported in future studies with larger groups of cases.