Abstract: In order to investigate the prevalence of Ascaridia galli in Huaihua City, Hunan Province, and to explore its genetic diversity and to elaborate the relationship of its phylogenetic development, in the experiment, a total of 430 chicken manure samples were collected from different areas of Huaihua City, Hunan Province, and the eggs were detected by saturated brine floating. IBM SPSS Statistics 22 software was used to collect and analyze the data, and the chi-square method was used to test the differences of Ascaridia galli infection in different areas and types of chicken. Some sequences of cox1 and nad4 genes were amplified and sequenced by PCR; the base composition and genetic diversity of the two genes were analyzed by Clustal X and DNASTAR software, and the phylogenetic relationship between Ascaridia galli was analyzed by Clustal X and PhyML 3.0 software. The results showed that the total infection rate of Ascaridia galli in Huaihua area of Hunan Province was 56.98%, including 9.07% in captive chickens and 47.91% in free-range chickens. There were some differences in the infection of Ascaridia galli in different areas and types of chickens. The length of the cox1 gene sequence was 953 bp, and the contents of four bases A, T, G, and C were 19.0%-19.3%, 45.9%-46.5%, 23.0%-23.2%, and 11.3%-12.0%, respectively; and the contents of A+T and G+C were 64.9%-65.8% and 34.3%-35.2%, respectively; and the intraspecific differences were 0-2.1%, and the interspecific differences were 13.12%-14.48%, respectively. The length of nad4 gene sequence was 876 bp, and the contents of four bases A, T, G, and C were 19.1%-19.4%, 52.4%-52.9%, 19.9%-20.2%, and 7.9%-8.3%, respectively; the contents of A+T and G+C were 71.5%-72.3% and 27.8%-28.5%, respectively; the intraspecific differences were 0-2.0%, and the interspecific differences were 26.37%-27.40%, respectively. Ascaridia galli from Huaihua area of Hunan Province was located on a branch of the phylogenetic tree. The results indicated that the epidemic of Ascaridia galli was serious in Huaihua City, Hunan Province, and the cox1 and nad4 gene sequences were AT preferred, and both of them had the characteristics of small intraspecific differences and large interspecific differences, which were good genetic markers. Although there was a certain degree of genetic variation and genetic diversity among their individuals, they were closely related.