Abstract: The Penaeidae comprises 26 genera and approximately 200 species as most of Penaeidae shrimps in the same genus are morphologically similar, and just exhibited subtle morphological differences, which makes 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 to 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 greater than the mean distance within-species. The phylogeny showed that 30 kinds of Penaeidae from 32 kinds of Penaeidae could converge upon a monophyly with high support values. We can see the good applicability of mitochondrial COⅠ-based DNA barcoding in Penaeidaes’ identification and use it as a necessary complement and a test of morphological taxonomic system.