Environmental conditions, such as temperature, carbon dioxide, and nutrient availability, are altered by urban conditions at regional scales with potential for impact on tree leaf structure. Our goal was to compare leaf morphological characteristics driven by physiological acclimation in red maple (Acer rubrum L.) trees in deciduous forests embedded in a small (Newark, DE) and a large (Philadelphia, PA) city. The study was conducted in six urban forests on eighteen mature red maple trees in a long-term urban forest network. We hypothesized that red maples in Philadelphia forests compared to Newark forests will have a thicker upper epidermal layer, spongy palisade and mesophyll layer, longer and wider stomates, and lower stomate density. Additionally, we hypothesized that red maples in Philadelphia forests compared to Newark forests will have lower leaf water content and specific leaf area, and greater leaf thickness, fresh leaf weight, dry leaf weight, and leaf dry matter content. Our results for stomate length and stomate width supported our predictions; red maple leaves had longer and wider stomates in Philadelphia forests than in Newark forests. The increased stomate size in red maple trees suggests potential altered gas exchange behavior and mutual abiotic stress mitigation responses in red maple to greater urbanization impacts in Philadelphia forests. This supports previous findings of possible physiological and biochemical acclimation of red maple trees to urban conditions. Furthermore, the findings from this study suggest red maple trees may be a good biomonitor of regional scale impacts in urban environments.