To better understand the effects of conjugated linoleic acid (CLA) administered at the early stages of embryonic development in poultry, it is necessary to analyze the molecular background. The molecular interpretation of poultry muscle after in-ovo administration of CLA remains to be reported. The purpose of the present study was to analyze the molecular background of muscle development based on gene expression parameters. On embryonic day (E) 11, the eggs were randomly divided into two groups (20 eggs per replicate). The CLA group was injected with 4.5 mg CLA in 100 mL of ethanol. The control group was injected with 100 mL of ethanol. At hatch, breast muscle samples were collected from four individuals per group for RNA-Seq. The result showed that 278 genes were differentially expressed (p<0.01, fold change >2) between the control group and the CLA group. 112 genes were upregulated in the CLA group, while 166 genes were upregulated in the control group. Function annotation showed that muscle activity (Ventricular cardiac muscle tissue morphogenesis, Adrenergic signaling in cardiomyocytes, Cardiac muscle contraction) and metabolism (Aromatic amino acid family metabolic process, Tyrosine metabolism, Retinol metabolism, Nitrogen metabolism, Cysteine, and methionine metabolism) were significantly enriched, including 11 genes(such as TNNI1, TNNC1, and MYL3) that were considerably upregulated in CLA group likely related to CLA processing by influencing the muscle activity processes and metabolic processes. It was concluded that in ovo, CLA supplementation could change the gene expression pattern of the breast muscle, and might affect muscle development.