Visfatin, an adipokine hormone produced primarily by visceral adipose tissue in mammals, has been implicated in the immune system, cellular aging, and glucose metabolism. Increased visceral adiposity and hyperglycemia have been correlated with elevated plasma visfatin levels in humans. The present study investigated visfatin cDNA and protein expressions, as well as plasma visfatin levels, in chickens that are selected for rapid growth and are naturally hyperglycemic relative to mammals. By RT-PCR, we detected visfatin cDNA in multiple tissues in the chicken. The deduced amino acid sequence of full-length chicken visfatin was 92–93% homologous to mammalian visfatin. Using real-time quantitative PCR and western blotting, chicken skeletal muscle was found to contain 5-fold and 3-fold greater quantities of visfatin mRNA and protein than abdominal fat pad, respectively. Visfatin mRNA and protein quantities were not significantly different among subcutaneous and visceral adipose tissue depots. Skeletal muscle visfatin mRNA and protein quantities, as well as plasma visfatin levels determined by enzyme immunoassay, were significantly higher in 8 week-old compared to 4 week-old chickens, possibly due to rapid skeletal muscle growth and visceral fat accretion occurring in broiler chickens during this period. However, fasting and re-feeding did not affect plasma visfatin levels in the chicken. Collectively, our results provide novel evidence that skeletal muscle, not the visceral adipose tissue, is the primary source of visfatin in chickens, thereby raising the possibility that visfatin may be acting as a myokine affecting skeletal muscle growth and metabolism.