The monogenean genus Heterobothrium (Monogenea: Diclidophoridae) currently consists of 12 accepted species described globally from various pufferfishes (Tetraodontidae). This includes the economically important Heterobothrium okamotoi Ogawa 1991 that causes severe disease in the cultured tiger puffer Takifugu rubripes in Japan. During parasitological surveys targeting near shore marine fishes of South Africa, a new Heterobothrium was discovered on the gills of five evileye pufferfish Amblyrhynchotes honckenii with a prevalence of 100% and mean intensity of 23 (4-72). Specimens were morphologically studied using both light and scanning electron microscopy and molecularly characterized by sequencing the partial ribosomal gene 28S. Heterobothrium victorwepeneri n. sp. differs from its 12 congeners by a combination of morphological characters, such as fourth clamp pair (anteriormost, 180° inverted) as the smallest with some of the sclerites differing in shape from the other pairs, 8 to 9 genital hooks, number of testes 40-50, and absent isthmus. The 28S tree depicted two main branches, one clustering together species of mazocraeidean families while the other clustered together only species of the Diclidophoridae. Heterobothrium victorwepeneri n. sp. is the first species of its genus to be recorded and described from South Africa and from the tetraodontid A. honckenii. This study also provides for the first time 28S sequence for a species of this highly host-specific genus.