Abstract: Previous studies have shown that geographical isolation, host specificity, and infection site are key factors shaping the genetic structure and differentiation of myxosporean populations; however, the magnitude and direction of their effects are species-dependent. To date, no such investigation has been conducted on Myxobolus pronini Liu et al. 2016. In the present study, we collected 19strains (S1—S19) of M. pronini from multiple localities to investigate whether the factors mentioned above have contributed to the population genetic structure and differentiation of this species, using 18S rDNA and ITS rDNA sequences as molecular markers. Morphological characterization demonstrated that spores from all 19strains conformed to the description of type material. Mature spores were elongated obovate in valvular view, with the blunt anterior end noticeably wider than the posterior end. Taking S1 as an example, mature spores measured (14.2±0.8) μm (13.3—16.4 μm) in length and (9.3±0.6) μm (7.9—9.9 μm) in width. The two pyriform polar capsules were equal in size, measuring (5.2±0.3) μm (4.7—6.0 μm) in length and (3.1±0.3) μm (2.4—3.5 μm) in width, with polar filaments coiled 5—6 turns. Pairwise comparisons of 18S rDNA sequences among 23strains (including 19 new and 4 previously published) revealed 99.7%—100.0% similarity and genetic distances of 0.0000—0.0027. The Russian strain (S22) exhibited relatively lower sequence similarity (99.7%—99.9%) and higher genetic distance (0.0014—0.0027) compared with the highly similar Chinese strains (99.9%—100.0%). For ITS rDNA, sequences similarity ranged from 99.4% to 100.0% and genetic distances of 0.0000—0.0055, indicating slightly higher intraspecific variation than that in 18S rDNA and suggesting ITS rDNA is more sensitive in detecting population-level differences. Phylogenetic analyses based on both 18S rDNA and ITS rDNA did not recover any geographically distinct monophyletic clusters or reveal any obvious association with host species or infection site, indicating that none of these factors has yet driven substantial population genetic differentiation in M. pronini. Although slight genetic variation was detected among geographic strains, the overall level of genetic diversity remained low, implying that geographical isolation exerts only a limited influence on population genetic differentiation in this species.