Myxosporeans are a group of microscopic parasites that primarily parasitize fish with more than 2600 species currently discovered worldwide. The rich diversity of this group has led to extensive research on population divergence in myxosporeans. To investigate the impacts of infection site differentiation on the population divergence of myxosporeans, Thelohanellus kitauei Egusa & Nakajima, 1981 was isolated from the skin (S-type) and intestine (I-type) of different common carps Cyprinus carpio L. We compared the morphological differences in the T. kitauei found at the two infection sites by measuring the morphological characteristics of both plasmodia and spores. Tissue samples from the infected skin and intestine were examined by histopathology. The small subunit (SSU) ribosomal DNA (rDNA) and internal transcribed spacer (ITS)-5.8S rDNA sequences of two isolates were also amplified, aligned, and phylogenetically analyzed with other T. kitauei sequences from GenBank. The results showed that the S-type formed a single plasmodium with a diameter of 2.1 cm, while the I-type formed sixteen plasmodia of different sizes (diameters of 0.21–0.82 cm). The membrane sheath widths of the S- and I-types were approximately identical (P＞0.05). However, the spore lengths, polar capsule, and membrane sheath of the S-type were significantly greater than those of the I-type (P＜0.001), while other morphological characteristics were significantly smaller than those of the I-type (spore thickness: P＜0.01; the width of spore and polar capsule: P＜0.001). Histologically, most of the S-type spores were distributed in the stratum spongiosum of the dermis, with some spores penetrating the stratum compactum, causing curvature and deformation of the dense connective tissue. The I-type spores were primarily scattered over the intestinal submucosa, and the intestinal villi squeezed by the spores showed atrophy to varying degrees. Both types infected the connective tissue, causing inflammatory reactions such as lymphocyte infiltration and tissue hyperplasia. Molecular sequence comparison showed that the SSU rDNA sequence similarity between the S-type and I-type was 99.7%, with 4 variation sites. However, the ITS-5.8S rDNA sequence similarity was only 97.3%, with a total of 20 variation sites. The phylogenetic tree was constructed based on SSU and ITS-5.8S rDNA sequences, respectivly, demonstrating that the sequences of T. kitauei were divided into two lineages, ‘intestine’ and ‘skin’ subclades. In conclusion, the populations of T. kitauei infecting the skin and intestine of common carp showed varying degrees of difference in morphology, parasitic characteristics, and genetic information, providing obvious evidence of population divergence. It was demonstrated that the infection site differentiation is an important factor driving population divergence in myxosporeans.