BACKGROUND: Freshwater fish are affected with much parasitic diseases, among the most common are Henneguyosis caused by myxozoans of the genus Henneguya, which exhibit great diversity in fish from South America, particularly in the Brazilian Amazon. PURPOSE: In this present study, we describe the morphological and phylogenetic aspects of the small ribosomal subunit (SSU rDNA) of two new species of Henneguya infecting the gills from Hypophthalmus marginatus, a freshwater catfish from the Amazon. METHODS: In 148 specimens, has been observed cyst formation in different regions of the gills, intrafilamentary and intralamellar. These samples were collected for PCR amplification and phylogenetic analysis. RESULTS: The myxospores from each cyst have an elliptical spore body, consisting of two elliptical polar capsules, but differed in taxonomic morphometric measurements, such as total size, caudal length, spore body length and width, and polar capsule length and width. Phylogenetically, the species formed a clade with Henneguya spp. described in Siluriform fish in Brazil. CONCLUSION: These findings support the description of two species, Henneguya matosi n. sp. and H. marginatus n. sp., which infect different regions of the gills of Hypophthalmus marginatus, a commercially important catfish.