The evolution of parasites is often directly affected by the host’s environment or behavior. Studies on the evolution of the same parasites in different hosts are extremely attractive and highly relevant to our understanding of divergence and speciation. Here we analyzed the genetic variation of Equus roundworm populations in different hosts (horses, zebras and donkeys), and presented the first molecular evidence of divergence in Equus roundworms (Parascaris univalens). At the genetic level, Equus roundworms were mainly separated into two clades (Horse-derived and Zebra & Donkey-derived). This divergence began at 600-1500 years ago, which interestingly coincided with the domestication history of horses. We found that compared with horse-derived roundworms, most of the key enzymes related to glycolysis were under strong positive selection in zebra & donkey-derived roundworms, indicating that the evolution of the metabolic level was one of the main reasons for the divergence. In addition, we conducted a selective scan of resistance-related genes and found that the three populations were under different degrees of selection. This prompted us to pay attention to the possible impact of drugs on divergence, not just the drug resistance. This work supports that divergence or speciation is a continuous and dynamic process, and continuous monitoring of environmental factors is conducive to further understanding the adaptive evolution of roundworms.