The benefits and costs of amphistomy (AS) vs. hypostomy (HS) are not fully understood. Here, we quantify benefits of access of CO2 through stomata on the upper (adaxial) leaf surface, using 13C abundance in the adaxial and abaxial epicuticular wax. Additionally, a relationship between the distribution of stomata and epicuticular wax (EW) on the opposite leaf sides is studied. We suggest that the 13C content of long-chain aliphatic compounds of cuticular wax records the leaf internal CO2 concentration in chloroplasts adjacent to the adaxial and abaxial epidermes. This unique property stems from (i) wax synthesis being located exclusively in epidermal cells and (ii) ongoing wax renewal over the whole leaf lifespan. Compound-specific and bulk wax 13C abundance (δ) was related to amphistomy level (ASL, fraction of adaxial in all stomata) of four AS and five HS species grown under various levels of irradiance. The isotopic polarity of EW, i.e. the difference in abaxial and adaxial δ(δab-δad), was used to calculate the leaf dorsi-ventral CO2 gradient. Leaf-side specific EW deposition, amphiwaxy level (AWL), was estimated and related to ASL. In HS species, the CO2 concentration in the adaxial epidermis was lower than in the abaxial one independently of light conditions. In high-light and low-light grown AS leaves, the isotopic polarity and CO2 gradient varied in parallel with ASL. AS leaves grown under high light increased ASL compared to low light, and δab-δad approached near-zero values. Changes in ASL occurred concomitantly with changes in AWL. The leaf wax isotopic polarity is a newly identified leaf trait, distinguishing between hypo- and amphistomatous species and indicating that increased ASL in sun-exposed AS leaves reduces the CO2 gradient across the leaf mesophyll. Stomata and epicuticular wax deposition follow similar leaf-side patterning.