Using high‐resolution quadrupole time‐of‐flight mass spectrometry along with an electrospray ionization source (ESI‐QqTOF‐MS), accurate molecular weights of 13 steroid saponins extracted from the rhizomes of Dioscorea panthaica were acquired and the corresponding molecular formulae obtained. In order to elucidate the fragmentation pathways of steroid saponins in D. panthaica, 10 authentic samples were investigated using ESI‐QqTOF‐MS/MS. In addition, atmospheric pressure chemical ionization mass spectrometry combined with ion trap tandem mass spectrometry (APCI‐IT‐MS/MS) was used to analyze the structures of 13 steroid saponins in D. panthaica. Through the analysis of their tandem mass data, diagnostic fragment ions of the spirostanol and furostanol steroid saponins in D. panthaica were detected as m/z 271.2056 and 253.1951. In addition, four pairs of isomers were detected and the possible structures of four unknown steroid saponins in D. panthaica speculated. ESI‐TOF and APCI‐MSⁿ have proved to be effective tools for research on fragmentation mechanism of steroid saponins and the rapid determination of native steroid saponins in extract mixture, thereby avoiding tedious derivation and separation steps. Copyright © 2006 John Wiley & Sons, Ltd.