The equine Parascaris spp. is large, parasitic nematodes, and predominantly focuses on the intestine of foals and young weanlings. There are two roundworms, Parascaris equorum and Parascaris univalens, recognized among equine hosts. In this study, all fifty-nine Parascaris worms were harvested from three different equine hosts (twenty specimens from Equus zebra, twenty specimens from E. caballus, and nineteen specimens from E. asinus). The ribosomal gene (ITS) and mitochondrial genes (cox1 and nadh1) were amplified to identify and genetically characterize these worms. Analysis of ITS sequences revealed five genotypes among the fifty-nine worms, and the sequence similarity among the worms from E. zebra and E. caballus was at a high level (99.87%), while the one of E. asinus worms showed an apparent difference from the worms either from the E. zebra or from the E. caballus (sequence similarity ranging from 93.04 to 93.42%). Analysis of mitochondrial genes revealed that twenty-one (cox1 gene) and thirteen (nadh1 gene) unique haplotypes were defined among the fifty-nine worms. The shared haplotypes (four cox1 haplotypes and one nadh1 haplotype) only occurred between the worm populations from E. zebra and E. caballus. The cox1 and nadh1 haplotype sequences were respectively applied to construct phylogenetic trees. Although the topologies showed that E. asinus worm population had an obvious boundary with the worm populations of the E. zebra and the E. caballus, however, no noticeable boundary was found within the two later worm populations. Meanwhile, the E. asinus worm population showed an obvious genetic differentiation and an extremely low gene flow (close to zero) with the worm populations from E. zebra and E. caballus, indicating that the genetic characteristics of the worms from the E. asinus have an obvious difference with the one from E. zebra and E. caballus.