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Background: As a ubiquitous reversible epigenetic RNA modification, N6-methyladenosine (m6A) plays crucial regulatory roles in multiple biological pathways. However, its functional mechanisms in sex determination and differentiation during gonadal development of chicken embryos are not clear. Therefore, we established a transcriptome-wide m6A map in the female and male chicken left gonads of embryonic day 7 (E7) by methylated RNA immunoprecipitation sequencing (MeRIP-seq) to offer insight into the landscape of m6A methylation and investigate the post-transcriptional modification underlying gonadal differentiation. Results: The chicken embryonic gonadal transcriptome was extensively methylated. We found 15,191 and 16,111 m6A peaks in the female and male left gonads, respectively, which were mainly enriched in the coding sequence (CDS) and stop codon. Among these m6A peaks, we identified that 1013 and 751 were hypermethylated in females and males, respectively. These differential peaks covered 281 and 327 genes, such as BMP2, SMAD2, SOX9 and CYP19A1, which were primarily associated with development, morphogenesis and sex differentiation by functional enrichment. Further analysis revealed that the m6A methylation level was positively correlated with gene expression abundance. Furthermore, we found that YTHDC2 could regulate the expression of sex-related genes, especially HEMGN and SOX9, in male mesonephros/gonad mingle cells, which was verified by in vitro experiments, suggesting a regulatory role of m6A methylation in chicken gonad differentiation. Conclusions: This work provided a comprehensive m6A methylation profile of chicken embryonic gonads and revealed YTHDC2 as a key regulator responsible for sex differentiation. Our results contribute to a better understanding of epigenetic factors involved in chicken sex determination and differentiation and to promoting the future development of sex manipulation in poultry industry. [ABSTRACT FROM AUTHOR]