Knowledge of the leaf anatomy of grassland plants is crucial for understanding how these plants adapt to the environment. Tibetan alpine grasslands and Inner Mongolian temperate grasslands are two major grassland types in northern China. Tibetan alpine grasslands occur in high-altitude regions where the low temperatures limit plant growth. Inner Mongolian temperate grasslands are found in arid regions where moisture is the limiting factor. Few comparative studies concerning the leaf anatomy of grassland plants of the Tibetan Plateau and Inner Mongolian Plateau have been conducted. We examined leaf characteristics at 71 sites and among 65 species, across the alpine grasslands of the Tibetan Plateau and the temperate grasslands of the Inner Mongolian Plateau. We compared the leaf structures of plants with different life forms and taxonomies, and their adaptation to arid or cold environments. We explored relationships among leaf features and the effects of climatic factors (i.e., growing season temperature and precipitation) on leaf characteristics. Our results showed that (i) there were significant differences in leaf anatomy between Tibetan alpine and Inner Mongolian temperate grasslands. Except for mesophyll cell density, the values obtained for thickness of leaf tissue, surface area and volume of mesophyll cells were larger on the Tibetan Plateau than on the Inner Mongolian Plateau. (ii) Within the same family or genus, leaf anatomy showed significant differences between two regions, and trends were consistent with those of whole species. (iii) Leaf anatomy of woody and herbaceous plants also showed significant differences between the regions. Except for mesophyll cell density, the values obtained for the thickness of leaf tissue, and the surface area and volume of mesophyll cells were larger in herbaceous than in woody plants. (iv) Leaf anatomical traits changed accordingly. Total leaf thickness, thicknesses of lower and upper epidermal cells, and surface area and volume of mesophyll cells were positively correlated, while mesophyll cell density was negatively associated with those traits. (v) Growing season temperature had stronger effects on leaf anatomy than growing season precipitation. Although the communities in Tibetan and Inner Mongolian grasslands were similar in appearance, leaf anatomy differed; this was probably due to the combined effects of evolutionary adaptation of plants to environment and environmental stress induced by climatic factors.