The contents of sesquiterpene glycosides (SGs) from Dendrobium nobile Lindl. were greatly influenced by different growth conditions. A quantitative method based on HPLC‐QqQ‐MS/MS in MRM mode was developed for simultaneous analysis of six SGs. The results were further analyzed by principal component analysis and hierarchical cluster analysis. The results showed the SG contents were relatively high in 3‐year‐old D. nobile grown at an altitude of approximately 500 m on Danxia rocks in Chishui, Guizhou.