An HPLC method for determination of scoparone and ayapin was established for investigating the distributed patterns of scoparone and ayapin in 37 species of Dendrobium. The contents of scoparone and ayapin in varied collected samples were determined by the established HPLC method. The pseudo-bulbs sampled were collected according to different growth age of D. thyrsiflorum. The results showed that the contents of scoparone and ayapin were much differently distributed in species of Dendrobium. Only D. thyrsiflorum and D. densfilorum contained both scoparone and ayapin, the content decreased with the growth age. A fewer amount of ayapin was tested in D. loddigesii from Wenshan. The scoparone and ayapin were not determined in the rest species of Dendrobium. The method was concise, sensitive, accurate and reproducible. It could be applied to assay scoparone and ayapin in populations of herbal Dendrobium.