Digenetic trematode infections pose a significant threat to the African catfish aquaculture industry in Egypt. This study investigated the prevalence, morphological characteristics, molecular composition, and host responses to Prohemistomum vivax encysted metacercariae (EMC) infecting farmed African catfish (Clarias gariepinus). A total of 160 fish were collected from farms in Kafr-elsheikh Governorate between 2022 and 2023. Parasitological examination revealed an overall prevalence of 55% (n = 88) of infection with EMC of P. vivax. The prevalence of infection exhibited seasonal variations, with the highest rates in summer (100%), followed by spring (75%), autumn (30%), and winter (15%). Parasitic intensity showed significant major seasonal differences, with the lowest mean intensity in winter (1.6 ± 2.3 EMC per field) and the highest in summer (14.4± 9.3 EMC per field). Molecular sequencing and phylogenetic analysis of the internal transcribed spacer 2 (ITS2) gene unambiguously identified the metacercariae as P. vivax, overcoming limitations of traditional morphology-based techniques that cannot differentiate between digenean species when present as encysted stages. Histopathological examination revealed the presence of multiple parasitic cysts between hepatocytes, with diffuse vacuolar degeneration of hepatocytes. Muscles showed the presence of multiple parasitic cysts embedded in muscle bundles, with observed tissue reaction, inflammatory cell infiltration, oedema of interstitial tissue, and hyaline degeneration of muscle fibers. These findings highlight the importance of implementing effective control strategies to minimize the impact of P. vivax EMC infection on the Egyptian catfish aquaculture industry.