To explore and effectively identify the laboratory red crucian carp’s mitochondrial DNA barcode, we have identified the species of crucian carp. This research based on the laboratory red crucian carp and the other species of crucian carp (Dongting lake crucian carp, Japanese white crucian carp, Carassius auratus grandoculis, C. auratus langsdorfii, goldfish, goldfish-like fish and red crucian carp-like fish) as the test group, using Sanger sequencing for all individual mitochondrial COⅠ, COⅢ, Cytb, D-loop sequencing, and built the three gene splicing sequences, and analyzed their characteristics and ten kinds of sequence clustering characteristics of crucian carp molecular system evolution. The results showed that the splicing sequence of three genes (COⅠ, COⅢ, Cytb) and the evolutionary tree clustering of COⅠ gene can obviously find that ten different species of crucian carp can form different small branches, while the clustering of COⅢ gene and D-loop sequence can’t distinguish ten species of crucian carp at the same time. The results showed that COⅠ gene and the splicing sequences of three genes can effectively identify laboratory red crucian carp and other species of crucian carp, so this sequence can be used as an effective molecular marker of laboratory red crucian carp. Meanwhile, the evolutionary tree constructed by COⅠ, COⅢ, Cytb and D-loop gene can reflect the evolutionary relationship between ten different species of crucian carp.