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Two sibling species of Ascocotyle (Ascocotyle), Ascocotyle (A.) secunda sp. nov. and Ascocotyle (A.) tertia sp. nov., are described on the basis of their life cycles in Argentina. Both taxa use the hydrobiid snails Heleobia castellanosae and H. parchappei as the first intermediate host, and atheriniform fishes Cnesterodon decemmaculatus, Jenynsia lineata and Gambusia affinis as the second intermediate hosts; chicks served as experimental definitive hosts for both trematodes. Excysted metacercariae and adults of both species are morphologically indistinguishable, although some statistically significant differences were found in the following measurements of adults specimens: body length, ventral sucker length and width, the distance between the anterior extension of the vitellaria and the anterior border of the ventral sucker, and egg length. Everted gonotyls in vivo show differences in the size and number of papillae. Cercaria of Ascocotyle secunda can be differentiated from that of A. tertia in granulation and size of penetration glands, the presence or absence of a terminal spine, and lateral tegumental thickening of the tail. Metacercarial cysts differ in the thickness of cyst wall. Adults of both species are morphologically similar to A. (A.) tenuicollis and A. (A.) puertoricensis in possessing double rows of 32 circumoral spines (16 in each row), short intestinal caeca and a foliaceous, transversally oval gonotyl. However, they can be distinguished by their larval stages.