Abstract: In order to study the polymorphism of mstn gene in Japanese flounder (Paralichthys olivaceus) and develop new markers for its molecular assisted breeding, we srceened 120 individuals of Japanese flounder (Three double clone hybrid families: 60, three Gynogenesis families: 60) mstn genes for single nucleotide polymorphisms (SNP), and conducted multiple comparisons and SNPs markers validation on the growth traits with different genotypes. The results detected seven SNPs loci, all of which were transposition type mutation; three Exon regions (Exonl 1: G2063A; Exonl 3: C3883T, C4009A) were synonymous mutations; three Intron regions (Intron I: A2444T; Intron II: T3816C, A3832C); and one non Coding region at 3' end (3' UTR: C4564A). The correlation analysis between SNPs and growth traits show that A3832C and C4564A were significantly correlated with growth traits such as body mass, body length and body height (P<0.05). Among them, A3832C AA genotype and C4564A AC genotype showed advantages in growth; A3832C CC, C4564A AA and CC genotype showed disadvantages in growth, while the other SNPs loci had no significant impact on growth traits (P>0.05). A3832C and C4564A markers were validated in fast-growing population (F: 60) and slow-growing population (S: 60), and the validation results are consistent with the results of multiple alignments, indicating that the two SNPs loci have high stability. In summary, this study screened two marker loci (A3832C, C4564A) in the mstn gene of Japanese flounder, which are significantly related to growth traits, providing theoretical references for its molecular assisted breeding.