Compared to Chinese domesticated duck, Muscovy duck is the only species that retains the broodiness characteristic. Strong broodiness seriously limits its reproductive performance. In order to explore the molecular mechanisms that regulate broody behavior in Muscovy duck, this study used whole genome resequencing (WGRS) to obtain genomic variation sites of Muscovy ducks and conducted association analysis with broody traits. A total of 6,131,623 Single Nucleotide Polymorphisms (SNPs)were obtained from 295 female Muscovy ducks. After genome wide association study (GWAS) with the total broody days, average broody days, broody frequency and the first day of broodiness, 39, 130, 29 and 138 significant SNPs were obtained, respectively. The key genes annotated to these SNPs loci include NCOA6, MRPS22, SCAP, CRY2, CK1δ and EZH1, which could be candidate genes regulating the broodiness of Muscovy ducks. Functional analysis showed that over expression of MRPS22 upregulated the expression of CYP19A1 to promote the synthesis of intracellular estradiol, and downregulated the expression of CYP11A1 and 3β-HSD to inhibit the synthesis of progesterone to regulate broodiness of Muscovy ducks. The genetic polymorphism results showed that MRPS22: g.19000662G>A was significantly associated with average broody days. The average broody days in GA mutant ducks increased by an average of 2.23 days compared to wild GG type, which can be used for molecular marker for broody behavior selection. In conclusion, our study revealed MRPS22 regulated the broody performance by affecting the synthesis of estradiol and progesterone, and g.19000662G>A in MRPS22 was significantly associated with average broody days of Muscovy duck.