Native chickens are endangered genetic resources that are kept by farmers for different purposes. Native chickens distributed in a wide range of altitudes, have developed adaptive mechanisms to deal with hypoxia. For the first time, we report variants associated with high-altitude adaptation in Iranian native chickens by whole genome sequencing of lowland and highland chickens. We found that these adaptive variants are involved in DNA repair, organs development, immune response and histone binding. Amazingly, signature selection analysis demonstrated that differential variants are adaptive in response to hypoxia and are not due to other evolutionary pressures. Cellular component analysis of variants showed that mitochondrion is the most important organelle for hypoxia adaptation. A total of 50 variants was detected in mtDNA for highland and lowland chickens. High-altitude associated with variant discovery highlighted the importance of COX3, a gene involved in cell respiration, in hypoxia adaptation. The results of study suggest that MIR6644-2 is involved in hypoxia and high-altitude adaptations by regulation of embryo development. Finally, 3877 novel SNVs including the mtDNA ones, were submitted to EBI (PRJEB24944). Whole-genome sequencing and variant discovery of native chickens provided novel insights about adaptation mechanisms and highlights the importance of valuable genomic variants in chickens.