A new species of a microsporidium found in the freshwater teleost Gymnorhamphichthys rondoni, collected on the lower Amazon River, is described based on light, ultrastructural, and phylogenetic studies. This parasite develops in the skeletal muscle of the abdominal cavity, forming whitish cyst-like structures containing numerous spores. Mature spores, lightly pyriform to ellipsoidal with rounded ends and measuring 4.25 ± 0.38 × 2.37 ± 0.42 µm (n = 30), were observed. The spore wall, which measured about 102 nm, was composed of 2 layers with approximately the same thickness. The isofilar polar filament was coiled, with 9–10 (rarely 8) turns. The posterior vacuole appeared as a pale area, occupying about 1/3 of the spore length, and contained a spherical posterosome composed of granular material that was denser at the periphery. The myofibrils located near the spores appeared to be in advanced degradation. Molecular analysis of the rRNA genes, including the ITS region, and phylogenetic analyses using maximum parsimony, maximum likelihood, and Baysesian inference were performed. The ultrastructural characteristics of the spores and the phylogenetic data strongly suggested that it is a new species related to Kabatana, Microgemma, Potaspora, Spraguea, and Tetramicra. We named this new microsporidian from Amazonian fauna as Kabatana rondoni n. sp.