To study genomic diversity polymorphic DNA Markers are widely used as most of them are selectively neutral, more ubiquitous than polymorphic protein and enzyme markers. Mitochondrial DNA (mtDNA) characterized high mutation rates caused by lack of protective histones, inefficient DNA repair systems and continuous exposure to mutagenic effects of oxygen radicals. In the present study PCR-RFLP techniques were used to reveal polymorphism of mtDNA D-loop region in Bhotia and Buxa tribes of Uttarakhand. The results showed 3 restriction patterns for HaeIII enzyme for Bhotia community and no restriction pattern for Alu I enzyme. In Bhotia and Buxa tribes of Uttarakhand ethnic prejudice is very high and kinship marriages greatly occurred between them. Furthermore, both the tribes lived in a close geographical area and have a little and limited communication with other parts of the country and these conditions confirmed the low level of heteroplasmy and polymorphic patterns for mtDNA D-loop region that observed in this population.