Henneguya leporinicola is a parasite of the gill filament of Leporinus macrocephalus, a characiform fish belonging to the Anostomidae family, which is of major economic importance. Despite the damage it causes in fish, little is known about this parasite. Therefore, a study was undertaken with fourteen specimens of L. macrocephalus taken from fish farms in the state of Sao Paulo. The fish were collected and examined searching for lesions and/or myxosporean plasmodia. One of the specimens (7.14%) contained white elongated plasmodia in the gill filament. The mature spores had elongated bodies with polar capsules of equal size and a caudal length greater than body length. Morphological characteristics identified the parasite as H. leporinicola. Molecular analysis of the 18S rDNA sequence resulted in a 1954 bp, demonstrating significant genetic differences with previously described species of Henneguya/Myxobolus. Phylogenetic analysis comparing the 18S rDNA sequence of H. leporinicola with other species, previously described in South America, and the 20 closest species as indicated by BLASTn Max Score showed H. leporinicola as a basal branch of a subclade composed by Henneguya spp. parasite of characiform hosts.