Two species of heterophyid trematodes were found in the Magellanic penguin, Spheniscus magellanicus (Forster), from Patagonia, Argentina. Ascocotyle (Ascocotyle) patagoniensis Hernández-Orts et al. (2012) is re-described based on new, properly fixed specimens (original material from South American sea lion, Otaria flavescens Shaw, was from frozen hosts). Metacercariae of this species are reported and described for the first time from the heart of the silversides, Odontesthes argentinensis (Valenciennes) and O. smitti (Lahille), from Patagonia. Ascocotyle (Phagicola) cameliae n. sp. is described from the intestine of S. magellanicus. The new species is placed into the subgenus Phagicola Faust, 1920 because of the presence of a single row of circumoral spines and uterine loops and vitelline follicles being confined posterior to the ventral sucker. However, it differs distinctly from other members of this subgenus by the number (19–24) and length (23–31 μm) of massive circumoral spines and by the morphology of the ventrogenital sac with a large, simple gonotyl devoid of refractile bodies. Molecular data (partial 28S rDNA sequences) for both species are also provided. Matching sequences from metacercarial and adult stages helped elucidate partially the life-cycle of A. (A.) patagoniensis. The interspecific relationships and phylogenetic position of Ascocotyle were further assessed on a broad phylogeny on the Opisthorchioidea Looss, 1899. Ascocotyle (P.) ornamentata Shalaby, Esposito, Riegler & Carratù (1993) described from decomposed worms (all circumoral spines were detached) found in a dog in Egypt, with no type-specimens of this species deposited in a repository collection, is considered to be species inquirenda.