The fragmentation behaviors of the two types of modified pregnane glycosides from Cynanchum forrestii were investigated by positive ion electrospray ionization multi-stage tandem mass spectrometry equipped with an ion trap analyzer. The spectral data further illuminated the predominance of ESI-MS(n) technique on the identification of pregnane glycosides, especially of two sorts of modified pregnane glycosides with aglycone skeletons of 13,14:14,15-disecopregnane-type and 14,15-secopregnane-type, which differed in the presence of the characteristic [M-46+Na]+ ion. For sugar residues, the fragment ions were analyzed and some possible fragmentation pathways were proposed, especially for 3-demethyl-2-deoxythevetose, the glycosidic cleavage reaction was easier to occur than those of other sugar units in its moiety. The natures and differences of the pregnane cores, and the types and linked sequences of sugar residues were illustrated. According to these conclusions, eight new pregnane glycosides in the fraction of Cynanchum forrestii were structurally elucidated by HPLC/HRMS and HPLC-DAD/ESI-MS(n) techniques. Results of the present studies can benefit the rapid identification and structural determination of analogous constituents in crude plant extracts.