Cynanchum auriculatum and Cynanchum wilfordii are widely used as folk medicine in Eastern Asia. However, the indeterminacy in the authentic original plant material has resulted in the same appellative name being given to the two plants, and they are commonly misused. Therefore, it is necessary to establish an analytical method for discrimination as well as quality control of the two species. This study was to develop HPLC-UV methods for quality assessment of C. auriculatum and C. wilfordii and discrimination between the two species. Two HPLC methods to analyze eight marker compounds were established and validated. The first method analyzed seven marker compounds simultaneously on a reversed-phase column, while the second method analyzed a single marker compound, conduritol F, which exists only in C. wilfordii, on a Si-column. Thirty-nine batches of C. auriculatum and nineteen batches of C. wilfordii that were collected from different geographical regions of South Korea were analyzed by these methods. The constructed data matrix was subjected to principal components analysis and hierarchical cluster analysis in order to classify the samples. The established methods offer a potential strategy for authentication and differentiation of the two species.