Abstract: Trichodinids are tiny parasitic ciliated protozoans with high diversity, and mainly parasitic in the fishes, shellfishes and rarely in coelenterates. The serious infections caused by trichodinids may induce damage to the hosts, or even death in severe cases. Many previous studies focused on morphology and taxonomy of trichodinids have been carried out worldwide. Because it is difficult to differentiate similar species based solely on morphological characters, molecular examination has been accepted for identification of species. In recent years, some studies have begun to apply molecular approaches to shed light on the past confusion of identification of taxa. It has been a long time since Trichodinella myakkae (Mueller, 1937) Šrámek-HuŠek, 1953 was confused with its relative species, Trichodinella epizootica (Raabe, 1950) Šrámek-HuŠek, 1953 and Trichodinella subtili (Lom, 1959) Lom & Haldar, 1977. It is also difficult to distinguish T. myakkae from other Trichodinella species by the only morphological characters, for T. myakkae shares similar shape and size to the related Trichodinella species and is often mixed infection with these trichodinids which are difficult to be isolated individually. Moreover, T. myakkae was reported from different regions around the world and mainly isolated from freshwater fish hosts, such as Ctenopharyngodon idellus, Aplites salmoides and other fishes. Therefore, it is of great significance to study phylogeny and the influencing factors of intra—species differentiation. This study collected three host fishes of Hypophthalmichthys molitrix, Aristichthys nobilis and Pseudorasbora parva for the examination of trichodinids. Under a binocular dissecting microscope, individual trichodinid was isolated from living host fishes by glass micropipettes. Three strains of T. myakkae were obtained, and their photomicrographs of silver impregnated specimen were provided. The PCA results showed that the morphological characteristics of the three strains were highly overlapping on the scatter plot. Besides, five 18S rDNA and ITS-5.8S rDNA sequences were obtained, all of which are highly similar to T. myakkae (AY102176) (sequence similarity≥99.0%), and the GC content ranged from 50.8% to 51.2%. Taking T. myakkae (AY102176) as the reference sequence, based on 18S rDNA sequence, there were 7 variation sites in T. myakkae (AN) and T. myakkae (PP) respectively, and 8 variation sites in T. myakkae (HM). All strains appeared to have the same secondary structure, and the difference was only found in the primary sequence of V4 and V5 regions. The ML and BI trees constructed based on 18S rDNA indicated a highly similar topology. The results showed that there was no significant phenotypic difference among the three strains of T. myakkae, while T. myakkae showed the obvious difference from T. epizootica and T. subtili; meanwhile, T. myakkae (PP) showed the molecular difference from T. myakkae (HM) and T. myakkae (AN) at the intraspecies level. Based on the phylogenetic analysis, T. myakkae and T. epizootica were obviously independent, and all the strains of T. myakkae showed a significant coevolutionary relationship with their host fishes. In summary, our research clarified the confusions between T. myakkae and those similar or related species and provided the basic data for taxonomic and phylogenetic studies of Trichodinella species.