This study presents the de-novo assembly of the mitochondrial genome of the Bhadawari buffalo (Bubalus bubalis), a critically important livestock breed in India. Using next-generation sequencing (NGS) technology, we successfully assembled the complete mitochondrial genome and compared it to the mitochondrial genomes of other buffalo breeds, focusing on the Egyptian buffalo. Our analysis reveals a close phylogenetic relationship between Bhadawari and Egyptian buffalo, suggesting recent divergence. The findings of this study contribute to our understanding of the mitochondrial diversity within buffalo species and have implications for breed conservation, genetic improvement, and phylogenetic studies.