With abundant fungi species and rich biodiversity，Agaricales comprises a variety ofmedicinal，edible and poisonous fungi.To identify these fungi have important significance for the discovery of new medicinal edible fungi resources，and the prevention of mycotoxin hazard.Methods:In this study，50 species belong to 23 genus of 13 families in Agaricales were identified by DNA barcode.The internal transcribed spacer(ITS)were amplified and sequenced.Sequence lengths，base compositions were compared，and sequences results were analyzed using BLAST.A phylogenetic neighbor-joining(NJ)tree was also constructed for the identification of above-mentioned species.Results:The results indicated that the ITS sequence lengths of 50 fungi species ranged from 463 to 751 bp and the GC content intermediate between 37.1% and 59.4%.Finally，all 50 species were clearly identified by BLAST and NJ tree.It is concluded that the ITS region can rapidly and accurately identify Agaricales species.Conclusion:The proposed DNA barcode has great application value in fungi species identification.