Abstract: We investigated leaf variations under different environments to reveal the differences in leaf anatomical characteristics of Setaria viridis (L.) Beauv. among 15 populations from Jiangsu Province. Using paraffin sections and improved methods, microscopic observation and measurement were performed to determine the characteristics of the epidermis and transverse section of S. viridis leaves. Coefficient of variation, principal component analysis and cluster analysis showed that 17 anatomical characteristics in the leaf epidermis and transverse section from the different populations exhibited marked variations. Among these characteristics, thickness of midrib and abaxial epidermis showed the largest coefficient of variation. Principal component analysis indicated that thickness of leaf, height and width of parallel bundles, thickness of midrib, and height and width of midrib bundle were prominent in phenotypic variations. These structural characteristics showed the transporting and supporting capacity, features of stomata and anti-adversity ability of leaves. Cluster analysis demonstrated that the 15 different populations could be classified into three groups, which matched with the population distributions. Thickness of leaf, height and width of vascular bundles, thickness of bulliform cells and stomata density increased gradually among populations on the waterside and in alkaline land, wasteland and mountainous regions with decreasing stomata size, while drought resistance increased in order. These results suggested different variations under different environments, which is of great importance to the ecological adaptability of S. viridis.