Mining activities in the Athabasca oil sands region (AOSR) have contributed to an increase of polycyclic aromatic compounds (PACs) locally. However, many PACs found in the AOSR, and the combined effects of PAC mixtures have not been evaluated for genotoxicity in wildlife. Here, we examine whether mutation frequencies in AOSR river otters are correlated to PAC tissue burdens. We used single‐molecule polymerase chain reaction (SM‐PCR) to measure the mutant frequency of unstable DNA microsatellite loci in the bone marrow of wild river otters (n = 11) from the AOSR. Microsatellite mutation frequencies were regressed against liver PAC burden (total, low/high molecular weight [LMW/HMW], and parent/alkylated PACs), and to the distances from where the samples were collected to nearby bitumen upgraders. We found that microsatellite mutation frequency was positively correlated with total liver PAC burden. LMW and alkylated PACs were detected at higher levels and had a stronger positive relationship with mutation frequency than HMW (alkylated and parent) PACs. There were no significant relationships detected between mutation frequency and LMW parent PACs or the distance from bitumen upgraders. Furthermore, pyrogenic and petrogenic signatures suggest PACs in animals with high mutation frequencies were associated with combustion processes; although further investigation is warranted, due to limitations of diagnostic ratio determination with biotic models. Our findings support the hypothesis that PACs found in the AOSR increase mutation frequency in wildlife. Further investigation is required to determine if the elevated PAC levels associated with higher mutation frequency are due to natural exposure or elevated human activity.