Objective To control the quality of the species in Dioscorea L. better. Methods An HPLC-ELSD method was developed for the first time to simultaneously determine four bioactive ingredients:dioscin gracillin,protoneodioscin, and protoneogracillin in 31 samples belonging to seven species of Dioscorea L. from different areas. The column was an Inertsil HILIC (250mmx4.6 mm,5pm). The separation was carried out with a gradient program. The mobile phase was acetonitrile-water at a flow rate of 0.8 mL/min. Results The standard curve was rectilinear in the range of 0.464-12.97 gg (r=0.9969) for dioscin, 0.310-7.09 ltg (r = 0.9953) for gracillin, 0.469-11.66 gg (r=0.9970) for protoneodioscin, and 0.276-6.87 gg (r＝0.9992) for protoneogracillin. The recoveries of the markers were 98.1%, 100.1%, 97.2%, and 96.4%, respectively. The contents of the four components were quite different among the seven species of Dioscorea L. Conclusion The proposed HPLC-ELSD method is convenient,fast, accurate, and applicable for simultaneous analysis of multiple bioactive components of species in Dioscorea L.for quality control, which could facilitate discovering new natural resources of steroidal saponin.