Abstract: Trichodinid is regarded as one of microscopic parasitic ciliates with high diversity, which is mainly parasitic in fishes, mollusks, some tadpoles of amphibian, and minority of coelenterates in aquaculture. Some trichodinid infections can cause significant damage to their hosts. Trichodinids are diverse and distributed worldwide, with around 400 Trichodina species reported in different environments. However, despite advancements in molecular technology, the research on trichodinids lags behind other ciliate groups mainly due to insufficient or invalid molecular data in GenBank, which inhibited in depth studies. Trichodina hyperparasitis Chen & Hsieh, 1984, a freshwater trichodinid, parasitizes the crustacean Lampraglena chinensis, parasitizing on gills of Channa argus. Consequently, it was named T. hyperparasitis. Reports on T. hyperparasitis are fragmented, and molecular data are nearly blank, underscoring the need for additional valuable data to better understand this species, which is the focus of the present work. In order to explore the factors influencing population differentiation of T. hyperparasitis, three geographic strains were isolated from gills of Channa argus and Channa asiatica through extensive sampling. These strains include S strain from C. argus in Shapingba, Chongqing, D strain from C. argus in Dazu, Chongqing, and K strain from C. asiatica in Kaili, Guizhou. Among them, Guizhou represents a new geographic record, and C. asiatica is a new host record for T. hyperparasitis. Three SSU rDNA sequences from the three strains were sequenced and submitted to the GenBank, with further comparative study conducted based on morphological and molecular evidence. Morphological quantization of adhesive disc and PCA showed significant differences among the three geographic strains. The S strain was the largest in body size and denticle span (P<0.05), while K strain was the least one in denticle number (P<0.05). Intraspecific differences were also noted in adhesive disc structure and denticle morphology between both S and K strain (P<0.05). The comparative result of SSU rDNA sequences showed that ten base variant sites between K strain and the others, with a similarity range of 99.45% to 100% and genetic distance ranging from 0.001 to 0.003 among the three strains. The phylogenetic analysis based on SSU rDNA showed that T. hyperparasitis and its related species formed monophyletic lineages specific to host types, including the Perciformes and Siluriformes clades. Additionally, T. hyperparasitis formed host specific monophyletic lineages, viz. C. argus and C. asiatica clades. The comprehensive results indicate that T. hyperparasitis does not necessarily exhibit a hyperparasitic lifestyle and has a strong parasitic specificity to Channa species. Two strains from C. argus are closely related, while the strain from C. asiatica shows greater genetic and morphological divergence. This indicates that the host type, particularly initial habitat, may be the most direct factor influencing interspecific and intraspecific differentiation in ectoparasitic trichodinids.