Abstract: Based on its ultrastructural features of different developmental stages, molecular characteristics, and ecological considerations, the present study firstly discovered and identified Neoperezia semenovaiae Issi, et al. 2012 infecting the fat body of Chironomus plumosus larva in China. Transmission electron microscopy revealed numerous life stages of this microsporidian parasite. All developmental stages were monokaryotic and were in direct contact with the host cell cytoplasm. The early development stages observed were multinucleate merogonial plasmodia with high electron density. Later, merogonial plasmodia underwent plasmotomy to the formation of uninucleate or binucleate sporonts which further developed into sporoblasts. Sporoblasts were irregular in shape, surrounded by endoplasmic reticulum and transformed into mature spores. Mature spores were oval and monokaryotic, with a length of (5.7±0.2) μm(5.3—6.3 μm) and a width of (3.7±0.1) μm (3.4—4.0 μm). Spores possessed a bipartite polaroplast composed of an anterior spongy region and posterior lamellar membrane. Spore wall is trilaminar including an electron dense exospore measuring 26.5—62.7 nm, an electron lucent endospore with a thick of 151.8—236.1 nm, and a thin plasma membrane. Polar filaments were isofilar, coiled with 31—33 turns and arranged in 2 or 3 rows. The obtained sequences from amplification of the partial SSU rDNA was 1356 bp in length, which is 99.1% similarity to N. semenovaiae. Phylogenetic analysis based on the aligned partial SSU rDNA sequences indicated that N. semenovaiae clustered with species of Neoperezia, Bryonosema and Schroedera and this microsporidian parasite had undergone obvious genetic geographical differentiation.