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The myostatin (MSTN) gene, as a negative regulator of skeletal muscle growth, has been proposed to be associated with production traits in farm animals. In the present study, a T/C variant at -125 bp (relative to ATG start codon) of 5’regulatory region of rabbit MSTN was identified by direct sequencing. Two hundred and twenty two rabbits, which were randomly sampled from 3 breeds (Ira rabbits, Champagne rabbits and Tianfu black rabbits), were genotyped by high-resolution melting (HRM). Comparing the genotyping results of 47 samples with direct sequencing, the HRM showed high sensitivity (0.96) and high specificity (0.98). In the three rabbit breeds, the allele C was the predominant allele. The polymorphic site showed high heterozygosity (He = 0.48) and high effective number of alleles (Ne = 1.91). The genetic diversity was reasonably informative (0.25<PIC<0.50). The association analysis showed that the genotype TC had significant effect on the 84-d-weight of rabbits compared with genotype CC (p = 0.047). In contrast, the genotypes had no significant effect on other production traits. These results showed that HRM could be effectively used for genotyping analysis of MSTN gene. The T/C variant in 5’regulatory region of MSTN might be one of the candidate SNP loci affecting the trait of 84-d-weight.