BackgroundThe roundworms,Parascarisspp., are important nematode parasites of foals and were historically model organisms in the field of cell biology, leading to many important discoveries. According to karyotype, ascarids inEquusare commonly divided intoParascaris univalens(2n= 2) andParascaris equorum(2n= 4).MethodsHere, we performed morphological identification, karyotyping and sequencing of roundworms from three different hosts (horses, zebras and donkeys). Phylogenetic analysis was performed to study the divergence of these ascarids based on cytochrome c oxidase subunit I (COI) and internal transcribed spacer (ITS) sequences.ResultsKaryotyping, performed on eggs recovered from worms of three differentEquushosts in China, showed two different karyotypes (2n= 2 inP. univalenscollected from horses and zebras; 2n= 6 inParascarissp. collected from donkeys). There are some differences in the terminal part of the spicula betweenP. univalens(concave) andParascarissp. (rounded). Additionally, it was found that the egg’s chitinous layer was significantly thicker inParascarissp. (> 5 μm) thanP. univalens(< 5 μm) (F(2537)= 1967,P< 0.01). Phylogenetic trees showed that the sequences ofParascarisfromEquushosts were divided into two distinct lineages based on sequences of theCOIand ITS.ConclusionsComparing the differences in roundworms collected from three differentEquushosts, this study describes aParascarisspecies (Parascarissp.) with six chromosomes in donkeys. It is worth noting that the thickness of the chitinous layer in theParascarisegg may serve as a diagnostic indicator to distinguish the two roundworms (P. univalensandParascarissp.). TheParascarissp. with six chromosomes in donkeys in the present study may be a species ofP. trivalensdescribed in 1934, but the possibility that it is a newParascarisspecies cannot be ruled out. Both karyotyping and molecular analysis are necessary to solve the taxonomic problems inParascarisspecies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05768-3.