The classical major histocompatibility complex class I (MHC I) plays a vital role in the immune system. In this study, we cloned and identified golden pompano (Trachinotus ovatus) MHC Iα (Trov-MHC Iα), which encodes 351 amino acid residues including a leader peptide, α1, α2, α3 domain, a transmembrane region and a cytoplasmic domain. Twenty six different sequences, which encoded various numbers of amino acid residues ranging from 348 to 354, were obtained from 12 individuals. Highly genetic polymorphism was found in the Trov-MHC Iα, especially in the α1 and α2 domains. Meanwhile, in the α1 and α2 domains, 21 positive selected positions were revealed by site models, indicating the diversity of Trov-MHC Iα may be mainly generated by positive selection. Moreover, quantitative real-time reverse transcription PCR and western blotting analyses demonstrated that Trov-MHC Iα was ubiquitously expressed in the nine tested tissues and more highly expressed in intestine, head kidney, gill, and spleen. In the head kidney and spleen, Trov-MHC Iα was significantly upregulated under LPS or poly I:C stimulation. The results of this study provide valuable insight into molecular polymorphism, evolutionary mechanism, expression and function of MHC Iα in the immune system of golden pompano.