To improve the taxonomic characteristics of Myxobolus ampullicapsulatus and clarify its relationship with of M. honghuensis, M. ampullicapsulatus was comprehensively redescribed by morphological, histological and molecular methods, and a systematic comparison was made with molecular marker of M. honghuensis. In the present study, M. ampullicapsulatus was characterized by round or oval, white plasmodia in gills of gibel carp Carassius auratus gibelio, measuring 1.2-1.4 mm in diameter. Myxospores were pear-shaped in front view with pointed anterior end and rounded posterior, measuring 17.3-19.6 μm in length, 7.4-9.9 μm in width. Two ampullaceous polar capsules were unequal in size, with the larger polar capsule measuring 6.4-9.7 μm ×2.1-3.3 μm and the smaller one 5.3-8.9 μm ×2.0-3.3 μm. Polar filament coiled 8-11 turns. Histological analysis demonstrated that M. ampullicapsulatus developed within stratified epithelium between adjacent gill lamellae. A BLAST search conducted in this study revealed a high degree of similarity, ranging from 99.5% to 99.8%, between the obtained small subunit ribosomal DNA (SSU rDNA) sequence and those of M. ampullicapsulatus in GenBank (KC425223-KC425225, MH329620, JQ690361, JQ690373, KJ725082, MN227351, DQ339482). Furthermore, phylogenetic analysis indicated that M. ampullicapsulatus was closely related to M. honghuensis, serving as its sister species. Upon comparing the SSU rDNA sequences of M. ampullicapsulatus and M. honghuensis, it was observed that they shared a sequence similarity of 98.2% to 98.8%, a genetic distance of 0.014 to 0.018, and 27 base differences. The analysis of SSU rRNA sequences revealed that the secondary structures of different populations of M. ampullicapsulatus and M. honghuensis were found to be consistent, and there are obvious differences in the secondary structure between the two species. Moreover, notable differences were observed in the secondary structures of these two Myxobolus species, suggesting that the SSU rRNA secondary structure is expected to serve as a molecular feature to differentiate M. ampullicapsulatus and M. honghuensis. In summary, this study enhances the understanding of the detailed infection site of M. ampullicapsulatus in gills, and proposes that the SSU rRNA secondary structure may be used as an effective molecular marker for distinguishing M. ampullicapsulatus and M. honghuensis.