The aim of the study was to determine differences at the molecular level between different breeds of dogs on the basis of a comparative analysis of the mtDNA D-loop sequence. The material used in the study consisted of blood collected from 40 Doberman Pinscher, Dachshund, German Shepherd, and Crossbreed dogs. The investigations involved DNA extraction, amplification and sequencing of the mtDNA D-loop, as well as bioinformatic analyses. Fifteen SNP (single nucleotide polymorphism) mutations were identified. This study proposes a new solution for dog breed identification, i.e. the use of SNPs specific to each breed. The analysis revealed the presence of already known SNPs in various configurations depending on the breed. Breed-specific SNP patterns were identified in 94% of the Doberman dogs, and two SNP patterns were found in 73% of the German Shepherd dogs. This subject requires further research. However, given the limited amount of data on the use of mtDNA in determination of breed affiliation, the present results seem to be promising.