The intraspecific genetic diversity of freshwater fish inhabiting hydro-systems of the macrogeographic area spreading from the Black to Baltic Seas requires comprehensive investigation from fundamental and practical perspectives. The current study focused on the involvement of the mtDNA ATP6 region in the adaptability and microevolution of Perca fluviatilis within phylogeographic and anthropogenic contexts. We sequenced a 627 bp fragment encompassing the ATP6 region and used it for genetic analysis of 193 perch caught in Latvia, Lithuania, Belarus, and Ukraine, representing natural and anthropogenically impacted populations. We evaluated patterns of intraspecific genetic diversity in the ATP6 region and phylogeographic trends within the studied area compared with previously established D-loop trends. Evaluation of ATP6 coding sequence variability revealed that among 13 newly detected haplotypes, only two were caused by non-synonymous substitutions of amino acids of the protein. PCoA revealed three genetic groups (I–III) based on the ATP6 region that encompassed four previously described genetic groups established based on the mtDNA D-loop. The two mtDNA regions (D-loop and ATP6) have microevolved at least partially independently. Prolonged anthropogenic impacts may generate new point mutations at the ATP6 locus, but this phenomenon could be mainly concealed by natural selection and reparation processes.