Urban areas have globally expanded recently and will likely continue to do so in the near future. Although the impact of urbanization on acoustic and visual sexual signals has received considerable attention, other aspects, such as its influence on chemical signaling, remain poorly studied. Many lizard species possess femoral glands, i.e. prominent epidermal glands on the underside of the thighs producing a wide variety of compounds used in chemical signaling. Here we assessed the effect of urban, suburban and rural habitats and individual body condition on variation of chemical signal composition in the sand lizard (Lacerta agilis). By using gas chromatography coupled with mass spectrometry, we characterized chemical compounds present in secretions of lizard femoral glands. We found that lizards from suburban and urban habitats had the highest diversity of chemicals, while rural habitats showed significantly lower compound diversity. Lizards from urban and suburban habitats had high amounts of several compounds, including α-tocopherol, an antioxidant molecule that may counterbalance the damaging effects of irradiation on other pheromones. Chemical signals may not only depend on habitat characteristics but individual traits, such as body condition, may also affect chemical compound diversity. Body condition did not significantly differ across habitats, and we did not find an association between body condition and chemical composition of femoral gland secretions. We argue that environmental differences (more extreme in cities) as well as other factors such as increased stress may shape the amounts and diversity of semiochemicals in sand lizards. Our study provides insight on how environmental conditions imposed by urban-rural gradients may modulate chemical communication in vertebrates.