Abstract: Allium macrostemon Bunge from 18 different habitats in 11 areas of Guizhou Province were taken as study materials. Their nrDNA ITS sequences were compared and analyzed, combined with ITS sequences from Wenchuan in Sichuan， Hanzhong in Shaanxi, China and Korea downloaded from GenBank. Results showed that the lengths of A. macrostemon Bunge sequences from the different areas in Guizhou ranged from 534 bp to 537 bp and the G+C content ranged from 50.5% to 51.1%, with an average content of 50.9% and 16 variable sites, including conversion between T-C, T-G, and A-G. Based on the sequences of A. monanthum Maxim. and other Allium species from GenBank, the plants could be divided into four groups using Bayesian and maximum parsimony methods, with A. macrostemon Bunge polys found on one branch and A. macrostemon Bunge considered to be a monophyletic group. The A. macrostemon Bunge from different areas could be divided into four branches and the phylogenetic relationships of A. macrostemon Bunge from Qingzhen in Guiyang, Pingzhai in Liupanshui, Pingba in Anshun, Xingren in southwest Guizhou, and Sinan in Tongren were found to be very close, indicating that the phylogenetic relationships of A. macrostemon Bunge from different habitats could be identified through nrDNA ITS sequences.