Parvalbumin (PV) is the major allergen of fish species. So far, research concerning fish allergy has been focused mainly on marine fish. China is the largest consumer and producer of freshwater fish in the world, and many people suffered from allergy when consuming freshwater fish. Therefore, figuring out the molecular characters of PV from freshwater fish would be important. In this study, four gene sequences of PV isoforms were amplified and cloned from crucian carp and mandarin fish by RT-PCR, with two genes from each species. All the four genes were 330 bp in length, encoding 109 amino acid residues. The deduced molecular weight of them was approximately 11.6 ku, and isoelectric points were 4.45-4.69. The existence of amino acid residues of Ala-14, Leu-16, Cys-19, Phe-67, Gln-69 and Thr-79 suggested that all the sequences belonged to β-PV. The identity between the two sequences of crucian carp was 80.73 %, and was 83.49 % in mandarin fish. The identity between PVs from different fish species was higher than 75 % and the sequences of Ca²⁺ binding sites were highly conserved. Phylogenetic tree based on some PV amino acid residues revealed that PVs are clustered into two major clades, teleost fish and other vertebrates. PVs of teleost were clustered into orthologous groups, suggesting duplication of the PV genes before the speciation of teleost while after the speciation of fish. The tertiary structures of these four PVs were constructed using SWISS-MODEL. They all contain three helix-loop-helix motifs named EF-hand. Two domains next to the C terminal were Ca²⁺ binding sites. These results may provide a molecular basis for further study on the allergen of fish PVs.