Twenty-two strains of Colletotrichum, representing eight species, were isolated from eight genera of Orchidaceae in Yunnan and Guizhou provinces in China. Fourteen strains were from lesions and are pathogens, four were from fallen/dead flowers or stems and four were isolated as endophytes. The strains are characterized through morphological studies and multilocus phylogenetic analysis (ACT, Tub 2, CAL, CHS I, GPDH and ITS). Colletotrichum orchidearum and the new species Colletotrichum karstii are described and illustrated, based on morphological characters and multilocus sequence data. Collections of the other Colletotrichum species are reported with notes.