Kranz anatomy and C4 vein pattern are required for C4 biochemical functioning in C4 plants; however, the evolutionary timing of anatomical and biochemical adaptations is unknown. From the genus Flaveria, 16 species (C3, C4, intermediates [C3–C4, C4‐like]) were analyzed, novel anatomical and vein pattern characters were analyzed and key anatomical differences among photosynthetic groups were highlighted. A stepwise acquisition of anatomical and vein pattern traits prior to derived biochemistry was outlined on the basis of the phylogeny of Flaveria. Increased vein density represents a potential “precondition” contributing to lower ratios of photosynthetic tissues (mesophyll, bundle sheath) and precedes further anatomical and biochemical modifications observed in derived C3–C4 intermediates. In derived Flaveria species, bundle sheath volume is modified through cell expansion, whereas mesophyll volume is altered through mesophyll cell expansion, reductions in the number of ground tissue layers, and increased vein density. Results demonstrated that key anatomical features of C4 plants are also required for C3–C4 biochemical intermediacy, and anatomical and biochemical alterations acquired during evolution of intermediacy may predispose a species for evolution of C4 photosynthesis. C4‐like species are similar to C4 species, demonstrating that Kranz anatomy is fully evolved before complete C4 biochemistry is achieved.