Abstract: Myxobolus acinosus Nie & Li, 1973, Myxobolus pseudoacinosus Guo, et al., 2018 and Myxobolus toyamai Kudo, 1917 presenting high similar morphology, same host species and parasitic site, are difficult to be distinguished. In the present study, we conducted species identification and phylogenetic analysis on these three myxobolids. The myxospores of M. acinosus were long grape-shaped with slightly narrow and curved anterior and blunt posterior in valvular view. They were lens-shaped in sutural view. The spores were (10.8±0.4) μm (9.3—11.9) μm in length and (6.2±0.4) μm (5.0—7.1) μm in width. Two polar capsules were unequal. The larger polar capsules were pyriform with (4.6±0.5) μm (3.1—5.4) μm long and (2.6±0.3) μm (1.8—3.2) μm wide and 5—6 turns of polar filaments; the smaller ones were clavate with (2.3±0.3) μm (1.7—2.9) μm long and (0.9±0.1) μm (0.7—1.3) μm wide and 2—3 turns of polar filaments. The myxospores of M. pseudoacinosus were eggplant-shaped with narrow and curved anterior and blunt posterior. The spores were (15.9±0.5) μm (15.1.8—17.0) μm in length and (5.5±0.5) μm (5.0—6.3) μm in width. Two polar capsules were unequal. The larger polar capsules were pyriform with (7.7±0.5) μm (6.7—8.7) μm long and (3.5±0.3) μm (3.0—4.1) μm wide and 7—8 turns of polar filaments, the smaller ones were clavate with (3.2±0.2) μm (2.5—3.5) μm long and (0.9±0.1) μm (0.7—1.0) μm wide and 2—3 turns of polar filaments. The myxospores of M. toyamai were eggplant-shaped with slightly narrow and curved anterior and broad and blunt posterior in valvular view. They were pyriform-shaped in sutural view. The spores were (15.3±0.6) μm (14.3—16.6) μm in length and (6.2±0.4) μm (5.0—7.0) μm in width. Two polar capsules were unequal. The larger polar capsules were pyriform with (5.6±0.4) μm (4.6—6.4) μm long and (2.9±0.2) μm (2.4—3.4) μm wide and 6—8 turns of polar filaments, the smaller ones were clavate with (3.0±0.3) μm (2.2—3.7) μm long and (0.9±0.1) μm (0.7—1.2) μm wide and 2—3 turns of polar filaments. The morphological comparison of the three species showed that there were significant morphological differences among them. The 18S rDNA sequence similarity, genetic distance and variable site of the 4 strains of M. acinosus were 100%, 0—0.001 and 0—1, respectively; and the 18S rDNA sequences of the 4 strains had the highest similarity (99.9%—100%), smallest genetic distance (0—0.002) and least variable sites (0—2) with M. acinosus (KX810022, KX810021) from GenBank. The 18S rDNA sequence of the strain of M. pseudoacinosus had the highest similarity (99.8%) with M. pseudoacinosus (KX586684, KX810019) from GenBank, and genetic distance and variable site of the three sequences were 0.002—0.007 and 4—8, respectively. The 18S rDNA sequences of the 2 strains of M. toyamai were identical and they had the highest similarity (99.5%—100%) with M. toyamai (LC010116, C010115, FJ710802, HQ338729) from GenBank. The genetic distance and variable site among the six sequences were 0.000—0.002 and 0—2, respectively. The 18S rDNA sequence similarity, genetic distance and variable site between M. acinosus and M. pseudoacinosus were 98.4%—98.8%, 0.013—0.020 and 23—26, respectively, and that of M. acinosus and M. toyamai were 96.1%—97.2%, 0.038—0.042 and 55—58, respectively, and that of M. pseudoacinosus and M. toyamai were 96.4%—97.6%, 0.033—0.040 and 46—63, respectively. The analysis of 18S rDNA sequences showed that M. acinosus had 15 key variable sites which could distinguish the species from M. pseudoacinosus and M. toyamai; M. pseudoacinosus had 5 key variable sites which could distinguish the species from M. acinosus and M. toyamai; M. toyamai had 33 key variable sites which could distinguish the species from M. acinosus and M. pseudoacinosus. The model E23-2 of V4 region in 18S rRNA secondary structure could distinguish M. acinosus from M. pseudoacinosus and M. toyamai, and the model H43 of V7 region could distinguish M. toyamai from M. acinosus and M. pseudoacinosus. All evidences above implied that each of the three myxobolids had the independent species characteristics both in morphology and genetics. The phylogenetic analysis showed that M. acinosus, M. pseudoacinosus and M. toyamai clustered into a recent-diverged clade indicating the close relationship.