Urbanization, as indicated by increased impervious cover, alters freshwater communities via factors associated with urban stream syndrome. We hypothesized that livebearing Western mosquitofish, Gambusa affinis, from populations varying in levels of urbanization would differ in behavior and cognitive performance. Urbanization of six streams was determined by the percent of impervious cover, agricultural development, and underdeveloped land within the surrounding watershed. This species is tolerant and native but found invasive world-wide. Female fish were collected from the streams and acclimated in the lab and then were placed individually in holding containers within a larger arena for behavioral observation. We recorded the individual’s boldness, exploration, and latency to leave the enclosure. We then placed fish in a separate container in groups of 4 to record shoaling behavior. For cognitive performance we tested individual females using a detour test design with a motivation fish held at the opposite end in a transparent container. We measured problem solving and motivation to reach the other female behind a clear barrier in this maze. Individuals that explore more or faster may have higher cognitive skills. The fish from the population with an intermediate level (21%) of urbanization were less likely to emerge and decreased motivation compared to the other populations. The fish from the least urbanized population (2%) shoaled the most. Our results indicate that urbanization affects fish motivation and cognitive behaviors in a non-linear fashion.