Freshwater fishes were sampled from different localities in the River Nile at Qena Governorate (Qus, Qift, Dandara, El-Trammsa, El-Maana, Dishna and Nag-Hammady). Investigation revealed that Trypanosoma were found parasitized blood of many fish species. Out of 581 blood smears examined, 143 revealed trypanosomes infection with a prevalence of 24.6%. The infected fishes are belonging to seven species and four families. Trypanosomes parasites were found belonged to three species with different infection rates: T. mukasai Van As et al. 2004 (28.3%); T. cyanophilum Mohamed, 1978 (52.9%) and T. cataractae Putz, 1972 (1.5%). A new Trypanosoma species was found infecting Ch. ruppelli fish with an infection rate of 1.96%. Histological sections showed the presence of the adult stage of T. mukasai in between liver cells.  Also T. mukasai had been examined using scanning electron microscope.