Understanding and enhancing switchgrass (Panicum virgatum L.) photosynthesis will help to improve yield and quality for bio-industrial applications on cellulosic biofuel production. In the present study, leaf anatomical traits and physiological characteristics related to photosynthetic capacity of both lowland and upland switchgrass were recorded from four varieties across the vegetative, elongation and reproductive growth stages. Compared with the upland varieties, the lowland switchgrass showed 37-59, 22-64 and 27-73% higher performance on height, stem and leaf over all three growth stages. Leaf anatomical traits indicated that the leaves of lowland varieties provided more space for carbon assimilation and transportation caused by enhanced cell proliferation with more bundles sheath cells and larger contact areas between the bundle sheath and mesophyll cells (CAMB), which lead to the 32-72% higher photosynthetic capacity found in the lowland varieties during vegetative and elongation growth. However, photosynthetic capacity became 22-51% higher in the upland varieties during the reproductive stage, which is attributed to more photosynthetic pigment. In conclusion, lowland varieties gain a photosynthetic advantage with enhanced bundle sheath cell proliferation, while the upland varieties preserved more photosynthetic pigments. Our study provides new insights for improving the yield in crops by enhancing photosynthesis with anatomical and physiological strategies.