This study is the first time to perform molecular characterization in indigenous chickens, Sakini, of Nepal for studying genetic diversity and its relationship with its assumed progenitors. The first 522 nucleotides of hypervariable I (HVI) segment of the D-loop from 33 individuals were PCR amplified and subsequently sequenced. Fourteen haplotypes out of 33 sequences were identified from 20 polymorphic sites. Haplotype (gene) diversity (Hd) is 0.813 with SD 0.065 and nucleotide diversity (Pi) is 0.00525 with SD 0.00091. The neighbour joining tree indicated that Red Jungle Fowl from India is the progenitor of the Nepalese Sakini chicken. NETWORK analysis revealed that it can be grouped into four distinct Haplogroups (A1, E1, E2, and E3) respectively. Seventeen individuals belonged to E1, eight to E3, seven to E2, and one to A1. The high mitochondrial D-loop diversity in Nepalese Sakini chicken with multiple maternal origins serves the scientific basis for the development of rational policies supporting conservation efforts and provides directions for future research for developing sustainable genetic improvement approaches.