AbstractThe identification of novel tetrodotoxin (TTX,1) analogues would significantly contribute to the elucidation of its biosynthetic pathway. In this study, the first C5–C10 directly bonded TTX analogues, 4,9‐anhydro‐10‐hemiketal‐5‐deoxyTTX (2) and 4,9‐anhydro‐8‐epi‐10‐hemiketal‐5,6,11‐trideoxyTTX (3), were found in the newtCynops ensicauda popeiby using a screening method involving HILIC‐LC–MS/MS focused on the fragment ions of TTX analogues, and their structures were elucidated by spectroscopic methods. Compound2was detected in a wide range of newt species, and the2and TTX contents of 22 newt specimens were correlated (rs=0.88). Based on these results and its structural features,2was predicted to serve as a precursor of TTX that would be directly converted into 4,9‐anhydroTTX (4) by Baeyer–Villiger‐like oxidation or via 4,9‐anhydro‐5‐deoxyTTX formed by cleavage of the C5–C10 bond. The bicyclic carbon skeletons of2and3suggested a possible monoterpene origin for TTX.