Myosin light chains are major components of fish muscle fibers, and they play an important role in the process of muscle growth and contraction. The mandarin fish has been recently becoming one of the most important aquaculture fish species in China because of its good meat quality and protein composition. To a better understanding of the muscle development and its genetic controls, we constructed a cDNA library of the fast skeletal muscles the mandarin fish, S. chuatsi and successfully isolated two myosin light chains genes, MLC1 and MLC3. The length of the MLC1 and MLC3 cDNA was 1237 bp and 1070 bp, predicated encoding protein of 192 and150 amino acids, respectively. Excepting the 42 amino acid residues of the N terminal of the MLC1, the homology of the deduced amino acids sequences of the MLC1 and MLC3 was 80.3%. The two alkaline chains included two conservative EF-hand structures analyzed by PROSITE tools. The homology of the second EF-hand structure was 100% between the two genes, except the first three amino acids. However, MLC3 in fish did not discover the N-terminal sequence that was specific signs of MLC3 in higher vertebrates. Homologous comparison of the amino acid sequences of MLC1 and MLC3 in S. chuatsi with those the other fish species revealed certain variances in amino acid composition and species-specific characterization. To investigate the relationship the gene expression and muscle formation and development, we applied a real-time PCR technique to assay the ontogenetic expression of the two genes, and the results confirmed that the MLC1 and MLC3 mRNA were first detected in gastrula stage and its expression gradually increased from the muscular effect stage to larval stage, especially the two genes were highly expressed in the muscle effect and larval stages, that suggested their biological functions related to muscle differentiation and muscle motility. Our study provided detail information of the two alkaline MLC gene structure and its function on muscle development in S. chuasti.