The evaluation of the genetic diversity of indigenous chickens and the tracing of their matrilineal origin need to be facilitated to protect and utilize the germplasm bank of quality poultry. Twelve indigenous chicken breeds from Guangdong Province and its adjacent regions were used for mitochondrial DNA (mtDNA) D-loop sequencing, and analyses of genetic distance and genetic relationship between breeds were conducted. We constructed a phylogenetic tree and median-joining network based on haplotypes. Sixty mutation sites were detected in 360 samples, and all of these were found to be in transition. Eighty-five haplotypes, belonging to haplogroups A, B, C, and E, were defined. B was the predominant haplogroup (187, 51.94%), followed by E (76, 21.11%). Haplogroups A, B, C, and E were distributed in all the 12 breeds. B02 and C01 were the predominant haplotypes (85, 23.61%; 48, 13.33%), which were shared by all twelve breeds; and E03 ranked third (35, 9.72%), and was absent in the Xinghua, Huanglang and Ningdu yellow chicken. Xinghua chickens occurred mainly in haplogroup B, while Huiyang bearded and Zhongshan shalan chickens were distributed mainly in E. The number of haplotypes of Huaixiang chicken was the largest, while the Zhongshan shalan chicken had the fewest. The genetic distance between Guangdong chicken breeds ranged from 0.012 to 0.015, with haplotype diversity and nucleotide diversity ranging from 0.805 ± 0.047 to 0.949 ± 0.026, and from 0.0102 ± 0.0017 to 0.0138 ± 0.0009, respectively. Eighty-five haplotypes were divided into clades A, B, C, and E in the phylogenetic tree and median-joining network. Similar patterns of geographical distribution of mtDNA haplotypes was found in the chicken from Guangdong Province and its adjacent regions. A neutral test indicated that Guangdong indigenous chickens have not sustained obvious population expansion. Our results suggested that Guangdong chickens have a desirable conservation status, as they carry high levels of genetic diversity, and demographic histories were influenced to some extent, by the gene flow of domestic chickens from adjacent provinces and northern China. The red junglefowls from Southeast Asia have an important genetic contribution to the indigenous chickens of Guangdong Province.