Atmospheric CO₂ was estimated to be approximately 10 times higher than present when primitive ferns started to evolve. On the other hands, angiosperms emerged under CO₂ levels of only two- to three-fold higher than the present. This implies that ferns and angiosperms evolved under different CO₂ levels. Previous studies showed that stomatal frequency, which strongly affects stomatal conductance, has been shown to track changes in CO₂ levels. Leaf internal anatomy strongly affects plant photosynthesis traits, especially mesophyll conductance. We hypothesized that differences in leaf internal anatomy between ferns and angiosperms, or within the fern species emerged at different period, is affected by the CO₂ levels at their emergence. We used more than 100 extant ferns to observe their leaf internal anatomy comprehensively using light microscope and transmission electron microscope. Although all ferns had chloroplast in the epidermal cells, they showed smaller development in mesophyll chloroplasts compared to angiosperms. Cell wall thickness in ferns tended to be thicker than seed plants. The relationship between leaf internal anatomy and photosynthesis traits showed that large cell wall thickness and low development of chloroplast caused lower mesophyll conductance of ferns than that of angiosperms.