Abstract: Total of 19 steroids fragmentation pathways were investigated by electrospray ionization tandem mass spectrometry(ESI-MSⁿ) .Those steroids were isolated from the stems of Marsdenia tenacissima，which collected from Yun Nan province. And the fragmentation pathways of tenacigenoside G，tenacigenoside B，tenacigenoside H were summarized detailly in positive modeM+Na⁺. Combining with the experiment of other 16 compounds, the data reveals the first loss position is C—11 and C—12, then glycosides of C—3 lose orderly. This helps deduce the structures of those compounds according their structural characteristics and loss fragmentations. It also provides the basis for rapid identification of such compounds.