In recent years, the study on ribosomal genes has always been a hot spot. However, only a few reports on ribosomal genes of fish were reported, especially about the ITS1 (internal transcribed spacer 1) pseudogene. In order to investigate the applicability of ITS1 as a molecular marker in species identification and the phylogenetic analysis in fishes, 11 species from five families, belonging to Perciformes, were selected as the representatives, including Latidae, Toxotidae, Rachycentridae, Xiphiidae and Carangidae. A total of 348 ITS1 sequences were obtained, and the length of these sequences ranged from 442 to 661 bp. Compared with the genetic characteristics, such as the length differences, number of variable sites, GC content, nucleotide diversity and haplotype diversity, the ITS1 sequences of 11 species showed obvious polymorphism within intra-species and inter-species. The unusual case was that a shorter sequence with 55 bp deletion was detected among the 71 clones of Rachycentron canadum. Based on the data of GC content, secondary structure and minimum free energy of the shorter sequence, this sequence was inferred as pseudogene. The neighbor joining tree was constructed based on ITS1 sequences of 11 species from five families with Remora australis and Echeneis naucrates as outgroup. The result showed that the ITS1 sequences of different individuals from each species gathered together, and the species from each genus also grouped to one clade. These indicated the ITS1could be used as the molecular marker for phylogenetic analysis at the genus level. At the family level, the Carangidae was divided into two clades, which was similar to the result of phylogenetic analysis based on the mitochondrial marker, but both were contradictory with the classification based on morphological characteristics. To a certain extent, the ITS1 plays a role of molecular marker for phylogenetic analysis at the family level. These results provide useful scientific information for further studies on ITS1 in fishes and contribute to polymorphism analyses of ribosomal RNA genes in teleostean fishes.