The genus Trypanosoma Gruby, 1843 encompasses unique, flagellate haemoparasites infecting all vertebrate classes globally (excluding Antarctica). While trypanosomes in terrestrial mammals are well-studied due to their medical and veterinary significance, those in fishes remain largely unexplored, with limited data on their life cycles and ecological roles. Furthermore, the phylogenetic relationships of numerous aquatic species are unresolved. This gap is notable in South Africa, a region with high marine fish biodiversity, yet only 2 documented marine trypanosome species, 1 in teleosts and 1 in elasmobranchs are known. Our research aims to bridge this knowledge gap for marine fish trypanosomes along South Africa’s southern coast. Blood samples were collected from 246 fishes spanning 23 species at Chintsa East, Tsitsikamma (Garden Route National Park), Boknes, Kariega River Estuary and Groot River West Estuary from 2020 to 2023. Giemsa-stained blood smears were screened for trypanosomes, which were morphologically characterized. Molecular analyses targeting the 18S rRNA gene region were conducted on blood samples positive for trypanosomes. Combined morphological and molecular evidence identified 4 Trypanosoma species: 1 known species, Trypanosoma nudigobii from the klipfish (Clinus superciliosus) and 3 new species: Trypanosoma sp. A from the prison goby (Caffrogobius gilchristi), Trypanosoma bakana n. sp. from the white steenbras (Lithognathus lithognathus) and Trypanosoma bokkom n. sp. from 5 mullet species [the grooved mullet (Chelon dumerili), South African mullet (Chelon richardsonii), striped mullet (Chelon tricuspidens), fl athead grey mullet (Mugil cephalus) and the freshwater mullet (Pseudomyxus capensis)].