The main objective of this study was to better understand the characteristics of ITS2 sequence polymorphism in Pleuronectidae species. We cloned and sequenced ITS2 fragments in 10 species of Pleuronectidae, and then performed further analyses, including analysis of polymorphic statistics, calculation of Kimura-2-parameter (K2P) genetic distances, prediction of secondary structure and minimum free energy, and determination of recombinants. Finally, the phylogenetic relationship among different species was constructed by Maximum likelihood (ML) and Bayesian Inference (BI) method with Bothus myriaster as outgroup species. A total of 310 sequences were obtained from 10 species of Pleuronectidae. Among 6 of 10 species, Clidoderma asperrima (419﹣420 bp) and Hippoglossoides elassodon (419﹣420 bp) have the lowest variation in length, comparing to Lepidopsetta polyxystra (447﹣452 bp) and Limanda aspera (457﹣463 bp) with 5 bp and 6 bp variations, and Cleisthenes pinetorum (452﹣462 bp) and Verasper variegatus (465﹣479 bp) with 10 bp and 14 bp variations. For other four species, the biggest length variation of each species is 14﹣32 bp, resulting in longer, shorter or recombinant sequence types that are classified as types A, B and R. The biggest length variation occurs in Pseudopleuronectes yokohamae (454﹣486 bp), then Ps. herzensteini (433﹣458 bp), Eopsetta grigorjewi (420﹣439 bp), and Platichthys stellatus (466﹣480 bp) (Table 1). The K2P genetic distances of intra-species ranges from 0.002 to 0.027, except the high values in Pl. stellatus and Ps. herzensteini, up to 0.043 and 0.053, respectively; while the genetic distance of inter-species ranges from 0.046 to 0.180 (Table 2). The GC contents of ITS2 in the 10 species range from 63.95% to 70.16% (Table 1). The secondary structure has a uniform loop structure with five branches HelixⅠ﹣Ⅴ, except for V. variegatus with Helix Ⅳ-a and Ⅳ-b (Fig. 2). The phylogenetic trees constructed based on ITS2 sequences of 10 Pleuronectidae species show that the different clones of each species are clustered together (Fig. 3). According to the ITS2 sequence polymorphism characteristics, these four species Ps. yokohamae, Ps. herzensteini, E. grigorjewi, and Pl. stellatus with different types may be evolved in non-concerted evolution, while the other six species in concerted evolution. Although the K2P genetic distance of intra-species has overlap with that of inter-species, ITS2 is considered to be applicable for species identification among these species. The results of this study will enrich the ITS2 data of flatfish, and further provide a scientific reference for the researches on ribosomal RNA polymorphism of teleostean fishes.