Abstract: This study combined quantitative morphological and molecular methods to investigate intraspecific differentiation of Trichodina hypsilepis Wellborn, 1967 collected from six geographic regions (Chongqing, Sichuan, Guangxi, Hubei, Guizhou, and Guangdong) and five host species (Lateolabrax japonicus, Misgurnus anguillicaudatus, Paramisgurnus dabryanus, Hypophthalmichthys molitrix, and Danio rerio) in China. Multi-dimensional quantitative morphological comparisons revealed the following: (1) Analysis based on geographic region_host combination showed that the GX_LJ population exhibited significantly higher values than other populations in most core morphological indices (P<0.001), while the HB_DR population showed significantly lower values (P<0.001), representing the two morphological extremes. The CQ_LJ population was generally lower than the GX_LJ population, but remained significantly larger than the intermediate groups (P<0.05). No significant differences were found among MA populations from different regions, the GZ_HM, or the SC_PD population (P>0.05). (2) By geographic region, the GX and HB populations were identified as the morphologically largest and smallest groups, respectively, while the CQ, GZ, SC, and GD populations were intermediate. (3) By host species, the LJ and DR populations represented the morphologically largest and smallest groups, respectively, with the HM, MA, and PD populations being intermediate. The results of principal component analysis (PCA) from three different dimensions were generally consistent: most populations of T. hypsilepis overlapped with each other, while a few showed a separate distribution, indicating varying degrees of intraspecific morphological differentiation among populations of this species. Further main effect analysis showed that the host factor had a stronger main effect on the morphological differentiation of the adhesive disc than the geographical factor, but the two factors tended to act synergistically. Consistent with molecular findings, varying degrees of genetic differentiation have occurred among populations of T. hypsilepis. The HB_DR population represents an early diverging group within this species, exhibiting notable morphological and genetic differentiation, yet it has not reached the level of independent speciation. For populations derived from loaches (MA and PD population) that have not shown obvious morphological differentiation, varying degrees of genetic differentiation have already occurred at the genetic level. In summary, based on the morphological, molecular, and host evidence, the present study has validated the species validity of T. hypsilepis, a taxon that has been controversial in taxonomic history.