Ascaridia galli (Nematoda: Ascaridiidae) is the most common intestinal roundworm of chickens and other birds with a worldwide distribution. Although A. galli has been extensively studied, knowledge of the genetic variation of this parasite in detail is still insufficient. The present study examined genetic variation in the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene among A. galli isolates (n = 26) from domestic chickens in Hunan Province, China. A portion of the cox1 (pcox1) gene was amplified by polymerase chain reaction separately from adult A. galli individuals and the amplicons were subjected to sequencing from both directions. The length of the sequences of pcox1 is 441 bp. Although the intra-specific sequence variation within A. galli is 0–7.7%, the inter-specific sequence differences among other members of the infraorder Ascaridomorpha were 11.4–18.9%. Phylogenetic analyses based on the maximum likelihood method using the sequences of pcox1 confirmed that all of the Ascaridia isolates were A. galli, and also resolved three distinct clades. Taken together, the findings suggest that A. galli may represent a complex of cryptic species. Our results provide an additional genetic marker for the management of A. galli in chickens and other birds.