MicroRNAs (miRNAs) are small non-coding RNA molecules that play key roles in the regulation of development processes of many tissues and organs at the post-transcriptional level. However, little is known about how they affect chicken gonadal development. We examined the expression of four miRNAs (miR-218, -200b, -196, and -206) in chicken embryonic gonads at embryonic days 3.5-6.5. Their target genes were predicted by miRDB, TargetScan and PicTar algorithms. The expression levels of these four miRNAs differed with sex to varying degrees; miR-200b was expressed at a significantly higher level in female gonads during the entire interval. The whole mount in situ hybridization result showed considerably higher expression of miR-200b in females than in males in E5.5 embryos. The miRNA target scanning results indicated several genes with functions in gonad development and gonad function. We conclude that miR-200b is involved in the regulation of gonad development and sexual differentiation of chicken embryos.