Animal biodiversity is essential for maintaining the functionality of local food systems and ensuring sustainable livelihoods. Starting in 2000, the Food and Agriculture Organization of the United Nations (F.A.O.) has drawn attention to the decline in cattle populations, including the Transylvanian Pinzgau breed from Romania. Renowned for its hardiness, adaptability, and enhanced resistance to diseases and climate change, the Transylvanian Pinzgau is regarded as an important genetic asset for advancing livestock production. As a result, tracking genetic diversity has become a key focus in breeding programs, particularly for small, endangered local populations that play a vital role in regional agro-ecological systems. This research paper sought to assess the genetic diversity of a group of 24 head of cattle from the Transylvania region by analyzing two mtDNA markers, cytochrome b and D-loop sequences, both widely recognized for their relevance and importance in the analysis of genetic diversity of cattle and phylogenetic studies. The findings, derived through statistical analysis of nucleotide sequences using specialized software, indicated that the analyzed cattle are part of the ancestral T haplogroup, with a direct lineage tracing back to Bos taurus. This information can aid in developing crossbreeding programs focused on conserving essential genetic resources, improving other cattle breeds, and protecting biodiversity.