The aim of this study was to validate insertions located in homopolymeric regions of the MT-CO1, MT-CO2, and MT-CO3 genes, which were identified as potential false positives following Oxford Nanopore Technology (ONT) sequencing of canine mitochondrial DNA (mtDNA). The analysis included 22 blood and tumor tissue samples collected from five dogs with primary, recurrent and metastatic tumors. Sanger sequencing was employed to verify these variants. In total, 41 positions in homopolymeric regions were identified, where false insertions were systematically reported. Sanger sequencing did not confirm the presence of any of the detected changes in the examined regions, definitively classifying all observed variants as technical artifacts resulting from the specifics of nanopore sequencing.