Abstract: The matK gene plays an important role as the only group Ⅱ intros maturase in the chloroplast. We carried out bioinformatics analysis of the matK gene from five species of Botrychium. We then discussed the differences in matK gene sequences and MATK protein amino acid sequences in phylogenetic development of Botrychium. Results showed that the full length of the matK gene of the five species ranged from 1500-1503 bp, with 153 variation sites. The encoding products were all unstable hydrophilic proteins without a transmembrane structure, and the secondary structures were mainly alpha helices and random coils. Phylogenetic results showed that the phylogenetic tree constructed from the matK gene sequence was suitable for the identification of species of Botrychium. This study enriches current molecular information on matK in Botrychium and establishes the application value of matK in the identification of Botrychium.