The stomach, which is characterized by acid peptic digestion in vertebrates, has been lost secondarily multiple times in the evolution of the teleost fishes. The Cypriniformes are largely seen as an agastric order; however, within the superfamily Cobitoidea, the closely related sister groups Nemacheilidae and Balitoridae have been identified as gastric families. The presence of these most recently diverged gastric families in an otherwise agastric clade indicates that either multiple (>2-3) loss events occurred with the Cyprinidae, Catostomidae and Cobitidae, or that gastric reinvention arose in a recent ancestor of the Nemacheilidae/Balitoridae sister clade. In the present study, the foregut regions of Cobitidae, Nemacheilidae/Balitoridae and the ancestral Botiidae family members were examined for the presence of gastric glands and gastric proton pump (Atp4a) α subunit expression by histology and immunohistochemistry respectively. Atp4a gene expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Gastric glands expressing apical H+/K+-ATPase α subunit and isolated partial sequences of atp4a, identified using degenerate primers showing clear orthology to other vertebrate atp4a sequences, were detected in representative species from Nemacheilidae/ Balitoridae and Botiidae, but not Cobitidae (Misgurnus anguillicaudatus). In summary, we provide evidence for an uninterrupted gastric evolutionary lineage in the Cobitoidea, making it highly improbable that the stomach was reinvented in the Nemacheilidae/Balitoridae clade consistent with Dollo's principle. These results also indicate that the gastric trait may be present elsewhere in the Cobitoidea.