The Penaeidae comprises 26 genera and approximately 200 species ，as most of Penaeidae shrimps in the same genus are morphologically similar ,just exhibited subtle morphological differences,which make identification of Penaeidae shrimps a difficult task based on taxonomic keys alone.In order to define the practicability of DNA barcoding in identification of Penaeidae shrimps, in this study,we used COI gene to examine 32 Kinds of Penaeidaes’Nucleotide composition、interspecific genetic distance and intraspeccific genetic distance,and build Neighbor-joining tree of 32 kinds of Penaeidae based on mtDNA COI gene. As a result,The COI sequences of 32 kinds of Penaeidae present base preference. On average, the content of A+T (61.50%) was significantly higher than that of G+C (38.50%). As calculated by Kimera-2-parameter model, the mean distance within-species was 0.003 ,the mean distance pairwise-species (0.468) is 156 times bigger than the mean distance within-species.The phylogeny showed that 30 kinds of Penaeidae from 32 kinds of Penaeidae can converge a monophyly with high support values.we can see the good use of mitochondrial COI-based DNA barcoding in Penaeidaes’ identification,we can use it as a necessary complement and a test of morphological taxonomic system.