Artificial light at night is a pervasive pollutant that impacts diverse biological systems in myriad ways. One of the ways in which light pollution impacts organisms is through the disruption of natural circadian cycles, as many animals use these cycles to time behaviors key to fitness. A challenge of addressing the impacts of light pollution on biological timing in a natural setting is that light pollution is generally found most strongly in urban areas, which contain a multitude of factors (light, noise, temperature) that could all impact behavioral timing, and it can be difficult to parse the effects of these factors. Our ability to generalize the effects of urbanization is also hampered by logistical limitations in examining landscape-level patterns, and have traditionally focused on single species, single population comparisons. Here we address these challenges by examining variation in the timing of nightly calling critical for reproductive fitness of two cricket species in 14 populations across a large natural landscape of multiple urban and rural habitats. Moreover, we quantify the elements of urbanization (temperature, light, and noise) that might influence these patterns in each habitat. Our results demonstrate that both species alter their calling timing in response to natural variation in light at night, noise, and temperature, but that this variation is masked in urban areas, with resulting impacts on calling that could have important fitness consequences. Light pollution, even present at low levels, is an important management challenge in our efforts to conserve urban biodiversity.