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DNA barcoding is an e ective and widely used tool in biodiversity researches. Mitochondrial DNA barcodes serve as valuable markers for species identi cation, exploring boundaries among taxa, detecting cryptic diversity and solving existing taxonomic problems. However, numerous studies have demonstrated that mtDNA-based phylogenies are not always congruent with phylogenies inferred from the nuclear DNA. This phenomenon is called mito-nuclear discordance. Several mecha- nisms have been shown to generate mito-nuclear discordance, such as incomplete lineage sorting, mitochondrial introgression, natural selection or genetic sweeps mediated by Wolbachia infection. During the complex investigation of the Palaearctic Lepidoptera we have found striking example of mito-nuclear discordance in the taxonomically well-studied species - Moorland clouded yellow, Colias palaeno (Linnaeus, 1761). We sequenced a standard barcoding region (the 658 bp fragment of COI gene) for 850 specimens that spans the whole species distribution range. Analysis of DNA barcodes revealed ve deeply diverged mitochondrial lineages, that were characteristic of the species level. These lineages occur in sympatry and recovered as separate entities on the phylogenetic tree, with minimum uncorrected pairwise distances of the COI gene among them ranged from 1,5% to 9%, which exceed typical interspeci c COI distances reported for butter ies. In contrast to these results, we have found that Colias palaeno is genetically homogenous with respect to studied nuclear genes. The recovered mitochondrial lineages did not correspond neither to the phenotypic traits, nor to the clustering of nuclear genes. Thus, we found no clear evidence of cryptic species within Colias palaeno. Further investigation revealed strong as- sociation of the mitochondrial clades with two di erent Wolbachia strains, supporting the hypothesis that the mito- nuclear discordance resulted from selection on the mitochondrial genome due to selective sweeps by Wolbachia strains. We conclude that Wolbachia infection has played a key role in the evolution of mitochondrial genomes of Colias palaeno.