The Japanese natricine snake Rhabdophis tigrinus sequesters cardiotonic steroids, bufadienolides (BDs), from ingested toads in the nuchal glands as defensive toxins. A previous study showed that R. tigrinus in captivity converts dietary BDs when it sequesters them. However, it is unknown whether the dietary BDs are actually converted and the modified products accumulated under natural conditions. It is also unknown to what extent the BD profile of ingested toads is reflected in that of the snake. We collected 123 snakes from throughout Japan, analyzed their BD profiles by liquid chromatography/mass spectrometry, and identified 15 BDs from R. tigrinus by nuclear magnetic resonance analyses. We also compared their BD profiles using hierarchical cluster analysis (HCA). HCA exhibited two main clusters associated with their collection locations: eastern and western regions of the Japanese main islands. These results, coupled with previous findings on the BDs of Japanese toads, suggest that 1) R. tigrinus converts toad-derived BDs into other compounds under natural conditions; 2) there are both universal and regionally-specific conversions of dietary BDs by R. tigrinus; and 3) geographic variation in toad BD profiles is partially reflected in the variation of snake BD profiles.