Abstract: Zschokkella nova Klokacěwa, 1914, (Myxosporea, Zschokkella), infects nearly 30 fish species, causing dilation of bile and pancreatic ducts and hepatic parenchyma atrophy. Klokacěwa's original and the other descriptions lack geographical distribution data and molecular characterization, resulting in an incomplete database, which is not useful to link morphological and molecular traits, and hindering accurate species identification. Previous studies have revealed significant genetic differences among isolates from different host species. In order to supplement morphological and molecular taxonomic data, expand geographic distribution records, analyze differentiation patterns, and further reveal phylogenetic relationships, this study provided the new morphological data of three strains of Z. nova from Carassius auratus in different geographical regions with their corresponding 18S rDNA sequences. Morphological, molecular, and phylogenetic analyses were conducted among the strains parasitizing C. auratus, Carassius gibelio, Ctenopharyngodon idella, and Sander lucioperca. Mature myxospores of the new C. auratus-derived strains exhibited blunt ends, spheroidal or oval valve views, and oval suture views. Two pear-shaped capsules were located at both ends of the myxospore, were equal in size, and were centrally symmetric on both sides of the suture plane. The morphological characteristics of the three newly obtained strains of Z. nova were consistent with those of the type strain. Principal component analysis showed no significant differences in morphological data among the three geographical strains. The new sequences (MT014013, PV077966, and PV077967) displayed high similarity (99.89%—99.95%), with 99.92%—100% similarity to a C. auratus-derived Z. nova sequence (MH766650), 99.61%—99.68% similarity to another C. auratus-derived sequence (DQ377690), a similarity of 97.55%—97.71% with C. idella-derived (DQ377688) and S. lucioperca-derived (GU471266) sequences. Genetic distance values between the three new sequences and other Z. nova sequences (DQ377688; GU471266) were 0.020 (vs. DQ377688) and 0.019 (vs. GU471266), with corresponding numbers of divergent bases being 43 and 14, respectively. Phylogenetic analysis showed that the clades of Z. nova parasitizing C. auratus and C. gibelio formed an obvious sister relationship, while the C. idella-derived strain diverged basally in Bayesian inference tree or formed an independent branch in Maximum Likelihood trees. Notably, the S. lucioperca-derived strain constituted a distinct evolutionary branch, genetically distant from all the above-mentioned strains. These results suggest that Z. nova with multiple host species has already shown differentiation, and there may be a phenomenon of cryptic species, providing important data support for subsequent population genetics research on this species. Strains parasitic on C. idella and S. lucioperca may represent different species. Chongqing represents a new geographic distribution record for Z. nova. This study enhances the morphological, molecular, and geographical distribution database for Z. nova.