For conservation of indigenous poultry genetic resources and their use as genetic material in poultry breeding programs, it is necessary to obtain accurate genetic knowledge about these animals. The aim of this study was to investigate the genetic and phylogenetic characteristics of Khazak native chicken of Sistan usingthe nucleotide sequence of mitochondrial cytochrome b. For this purpose, blood samples were collected from 20 Khazak native chickens of Sistan. After DNA extraction, an 864 bp fragment of cytochrome b of mitochondrial genome was amplified by primers. The amplified fragments were purified and sequenced. A total of five different haplotypes were determined based on three single nucleotide polymorphism sequences. The final sequences from each haplotype with 789 bp in length were obtained, including 26.37% of adenine, 13.31% of guanine, 36.50% of cytosine, and 23.82% of thymine. The results indicated that the Khazak native chicken of Sistan has the lowest genetic distance from Japanese and Chinese native chickens.