A new species of Anoectochilus (Orchidaceae) from Guangxi, China, A. zhongshanensis, is described here, which was identified based on phylogenetic studies adopting combined plastid markers (rbcL-matK-trnL-F), morphological observation and chemical analysis. Molecular phylogenetic results support the systematic status of A. zhongshanensis as a new species in Anoectochilus genus. Morphologically, this new species is similar to A. zhejiangensis and A. malipoensis, but differs by its characteristic labellum and column, including the hastate or scalpel-shaped lobes of epichile, forward curved and pinnately divided cristate lobes at both sides of the mesochile and inverted triangle column wings. Furthermore, HPLC-ELSD analysis of these three species revealed the interesting chemotaxonomic difference that the principle and characteristic lactone glycoside in this new species was kinsenoside, rather than its diastereoisomer, goodyeroside A, a major glycoside in A. zhejiangensis and A. malipoensis.