The present study proposes to characterize the parasites isolated during the initial phase of production in fish farms located in Mato Grosso do Sul in the central-western region of Brazil, using histopathology analysis and molecular techniques. A total of 340 hybrid surubim fish (Pseudoplatystoma reticulatum × P. corruscans) from four farms were examined during the co-feeding phase. Histopathology analysis showed that 10.9% (n = 37) of the fish were infected with parasites. Branchitis, lifting epithelium, hypertrophy of epithelial cells, lamellar fusion, aneurisms and infection in the bone tissue of the gill filament was observed. The parasite species was determined by amplification of the 18S rRNA gene followed by sequencing. The phylogenetic analysis of nucleotide sequences indicates a close relationship (99.6%) with Henneguya pseudoplatystoma reported to be infecting the hybrid Pseudoplatystoma. This study demonstrates the occurrence of H. pseudoplatystoma in hybrid surubim (P. reticulatum × P. corruscans) during the co-feeding phase in fish farms in Mato Grosso do Sul. Also, molecular techniques provide a faster and sensitive method to identify fish parasites, and may assist in the development of new management techniques aimed at improving the sanitary conditions contributing to the reduction of mortality rates in these animals.