The Tatra chamois is an endemic subspecies of Northern chamois, indigenous only in the Tatras, a wild animal genetic resource unique to Slovakia. The main goal of this study was to describe the distribution of mtDNA haplogroups in isolated populations inhabiting geographical areas in the Slovak part of the Tatras and the development of methodologies for reliable differentiation between the closely related Alpine and Tatra subspecies based on mitochondrial DNA (mtDNA) sequence variation. A partial sequence of the D-loop region was obtained to enable analysis of the mtDNA and subsequent estimation of the population haplotype structures. Despite the relatively short fragment of the D-loop region (final length 480 bp) analysed, reliable differentiation of the selected subspecies as genetically unique was obtained. Moreover, the study results indicated the occurrence of four haplotypes within the sampled population. Except for three previously published haplotypes, the analysis of the target mtDNA region suggested the occurrence of a new group, here noted as Tatra 10, indicating the existence of a new, unique haplotype. From the geographical point of view, this new haplotype, seems to be restricted to the area of the Western Tatras. Thus, our study provides new insight into the haplotype structure of the native Tatra chamois population in Slovakia and can be beneficial for the management of its conservation status. It should be emphasised that the Tatra chamois is regarded as a critically endangered species. Therefore, the systematic monitoring of genetic diversity, including the distribution of mtDNA haplotypes, is necessary to develop an appropriate conservation strategy.