AbstractCloning and sequencing of various tropomyosin isoforms expressed in chickens have been described since the early 1980s. However, to the best of our knowledge, this is the first report on the molecular characterization and the expression of the sarcomeric isoform of the TPM3 gene in cardiac and skeletal muscles from developing as well as adult chickens. Expression of TPM3α was performed by conventional RT‐PCR as well as qRT‐PCR using relative expression (by ΔCT as well as ΔΔCT methods) and by determining absolute copy number. The results employing all these methods show that the expression level of TPM3α is maximum in embryonic (10‐day/15‐day old) skeletal muscle and can barely be detected in both cardiac and skeletal muscles from the adult chicken. Our various RT‐PCR analyses suggest that the expression of high molecular weight TPM3 isoforms are regulated at the transcription level from the proximal promoter at the 5′‐end of the TPM3 gene.