This study was to investigate the genetic diversity and population structure of Huainan yellow partridge chickens. The complete D-loop region of mitochondrial DNA (mtDNA) of 32 chickens were sequenced and compared with published data of the red jungle fowl. The D-loop region of Huainan yellow partridge chickens was 1 231-1 232 bp in length. The average haplotype diversity, nucleotide diversity and nucleotide differences were 0.917, 0.006 6, and 7.573, respectively. A total of 14 haplotypes were identified, which belonged to four previously published clades, i.e. Clades A, B, C, and E, and contained 3, 3, 7, and 1 haplotypes, respectively. The results showed a high mitochondrial D-loop diversity and indicated multiple maternal origins of Huainan yellow partridge chickens. The results in this study will be a reference for conservation and utilization of Huainan yellow partridge chickens.