Objective This study aims to investigate the infection of Clonorchis sinensis (C. sinensis) in high-incidence areas of Hunan Province, China. The phylogenetic analysis of the C. sinensis species in the highly infected areas was carried out.Method Infection of the definitive human host and intermediate fish host by C. sinensis was investigated, and the mitochondrial genes cox1 and Nad1 were used as genetic markers for phylogenetic analysis. Results In 2016–2020, the average population infection rate of Hunan was 1.38％, while in Tongdao County the rate was up to 26.90％, and the highest fish infection rate was detected in Qiyang County (99.44％ in the dorsal fin of crucian carp). High genetic sequence similarity was observed in the samples from Qiyang and Lengshuitan which exhibited high homology with those from Guangdong and Gansu, whereas the parasitic species from Tongdao was highly homologous with those located in high-latitude areas. Moreover, no significant difference was found in the gene sequence of the parasitic species in definitive hosts dogs and cats. Conclusion The systematically study of C. sinensis infection in the high-incidence areas will contribute greatly to the prevention and effectively controlling the spread of Clonorchis sinensis in Hunan Province The endemic of C. sinensis infection in Hunan Province is the result of co-action of local and foreign parasite species.