The genome-wide DNA methylation assay was used to analyze the difference in methylation between the breeding and conservation populations of Shaoxing ducks. The methylation level of the breeding population was higher than that of the two conservation populations, and the proportion of CG methylation sites was the largest in the three populations, most of the methylation sites were located in the exon region. There were 1247 different methylation regions in the two populations (group A and B), and 927 different methylation regions in the two groups (group A and group C). The differential methylation regions of the three groups were evenly distributed in the gene and intergene regions. GO and KEGG enrichment analysis showed that the differentially expressed genes in the A and B groups were mainly involved in synaptic and cell connections and the signaling pathways were significantly enriched in cAMP and oxytocin signaling pathways. The results showed that the group C was significantly enriched in eight signaling pathways, including the cAMP signaling pathway and long-term enhancement, compared to the group A. There were thirty-five differentially methylated genes, including , , , , , , , , , , , and . These genes were involved in the development and ovulation of ovaries and follicles and were closely related to the excellent production performance of the breeding population. In addition, , , and other genes related to eggshell quality were identified, which can be used as molecular markers to improve eggshell quality in the future.