Abstract: Macromitrium in China has complex morphological variation and many taxonomical problems. DNA barcoding is a new species identification technology. Thirty-eight specimens belonging to Macromitrium gymnostomum Sull. & Lesq., M. ousine Broth., M.taiheizanense Nog., Macromitrium cavaleriei Card. & Thér., M. japonicum Dozy & Molk., M. rhacomitrioides Nog. M. microstomum (Hook. & Grev.) Schwgr., and Macrocoma tenue subsp. sullivantii and Schlotheimia grevilleana were collected from Zhejiang, Fujian, Yunnan, Guangxi and Sichuan Provinces. The sequences of four chloroplast genes (trnL, trnG, psbT and rps4) were sequenced. Fifteen Bayesian phylogenetic trees were made based on different combinations of the above chloroplast genes. Species identification rates, and genetic distances between and within species of Macromitrium in the present study were calculated. We found that combinations of trnL-rps4, trnL-trnG-rps4, trnL-psbT-rps4, trnG-psbT-rps4 and trnL-trnG-rps4-psbT could be applied to effectively identify Macromitrium species, with 100% species identification rate. Based on the successful amplification and sequencing rate, as well as the phylogenetic relationships among the seven species of Macromitrium, the trnL-trnG-psbT combination was recommended for the DNA barcoding of the genus Macromitrium.