The millet variety Huangjinmiao represents yellow leaves at the seedling stage and recovered green leaves at the later stages. In this study, we attempted to decipher the physiological and genetic basis of the yellow-leaf seedling phenotype of cv.Huangjinmiao. The leaf color of cv. Huangjinmiao from yellow to green was observed at the booting and heading stages. The dynamic analysis showed that the contents of both chlorophyll a and b at the seedling stage were lower in cv. Huangjinmiao if compared to those of the control plants. At the heading stage, the chlorophyll a became no detectable difference, whereas the chlorophyll bremained significantly lower in cv. Huangjinmiao. The chloroplast ultrastructure showed lower number of chloroplasts and stacked thylakoids, and lower thickness of the stacked thylakoids in cv. Huangjinmiao extensively at the seedling stage but at the heading stage.The accumulative results clearly indicated a compromised biosynthesis of chlorophyll that occurs in cv. Huangjinmiao. The net photosynthetic rate of cv. Huangjinmiao was statistically lower than that of the control lines. The segregation analysis sing a F2 segregation population suggested a recessive-inherited single gene which manipulates the yellow-seedling phenotype. The preliminary mapping suggested that the causal gene was localized on chromosome 8 of Setaria italica. In conclusion, this study laid a good foundation for future high-resolution mapping and functional analysis of the causal gene. Moreover, the application potential of the yellow-leaf color of cv. Huangjinmiao as a phenotypic marker in breeding has been discussed.