The diosgenin concentrations in a Dioscorea polygonoides tuber collection from the Eje Cafetero (EC, Colombia) were determined by HPLC and their percentages ranged from 0.02 to 2.64%. The highest diosgenin concentration (2.64%) was obtained in a tuber collected near Salento (Quindío). The average of diosgenin recovery was 97%. Diosgenin was identified by gas chromatography-mass spectrometry (GC-MS) and by coelution with authentic diosgenin standard in both HPLC and GC-MS techniques. The results of this study are important since comparatively they are higher than most of those reported on other medicinal Dioscorea species, making Dioscorea polygonoides a potential new source of diosgenin.