Phytochemical investigation of the chemical components of Stemona tuberosa led to the isolation of two new alkaloids named tuberostemonine K ( 1) and tuberospironine ( 2), together with the known tuberostemonine ( 3). The new structures of 1 and 2 were elucidated through extensive spectroscopic analyses, while the molecular structure of 3 was confirmed by X-ray analysis. A gradient reversed-phase HPLC-ELSD method was established for the investigation of the chemical diversity of S. tuberosa from 13 localities, and four types of chemical variation featured by the major components 3, neotuberostemonine ( 4), croomine ( 5), and stemoninine ( 6), respectively, were observed.