China is an important domestication center of yams, and two main yam species of Dioscorea opposita and D. alata are commonly cultivated in China. However, the differences of nutritional and medicinal characteristics between the two species and their subgroups remain unclear, which would greatly affect the resource conservation and commercial utilization of yams. In this study, typical yam resources including the species of D. opposita (wild and cultivated Ruichang yam from south China, and Tiegun yam from north China) and two landraces of D. alata (Longyan yam and Anyuan yam from south China) were selected as materials. Nutritional traits and medicinal characteristics were determined and analyzed respectively. The results showed that there was no significant differences in the content of most nutrients between D. opposita and D. alata, but most cultivated Ruichang yam of D. opposita showed higher levels of starch, soluble sugar, sucrose, and ascorbate in tuber than that in yam from D. alata. Moreover, an UPLC-MS method was developed for identification and determination of medicinal characteristics in the two species. The results showed that allantoin can be detected in all selected samples. Cultivated Ruichang yam of D. opposita possessed the highest allantoin content among the tested materials, and was significantly different with that in Tiegun yam and D. alata. Dioscin was not detected in D. alata. Overall, there was little difference in nutritional composition between D. opposita and D. alata, but the medicinal quality of D. opposita was better than that of D. alata. Due to the outstanding comprehensive quality, the local variety of cultivated Ruichang yam can be further developed and utilized.