For a long time, the ribosomal RNA gene was thought to conform to the paradigm of strict concerted evolution pattern. In fact, an increasing number of intraindividual and intraspecies variations have been discovered, indicating non-concerted evolution. In order to explore the polymorphism of the first internal transcribed spacer (ITS1) in the species of Soleidae in Pleuronectiformes and whether it is suitable as a marker for species identification, a total of 118 ITS1 sequences were determined from five soles. The results showed that two distinct types were found in Pardachirus pavoninus genome, suggesting a non-concerted evolution pattern, while concerted evolution was confirmed in other four soles because of absence of intraindividual polymorphism. Sequence analysis demonstrated that ITS1 had significant length heterogeneity, ranging from 412 bp in Zebrias quagga to 585 bp in P. pavoninus. The base content of sequences in all five soles was in the same trend with C > G > A > T and GC content (69.5%) was far higher than AT content. Except for one sequence of P. pavoninus, each of five soles clustered into one clade, respectively, suggesting ITS1 sequence was suitable for species identification in the present study. It is worth noting that the sequence of P. pavoninus clustered with Brachiru orientalis, suggesting that ITS1 sequence polymorphism interfered species identification, as a result, it is essential to obtain enough clones before using ITS1 sequence as the molecular marker for species identification in order to avoid incorrect conclusions.