Predators often avoid aposematic prey as a result of aversions to particular prey signals (e.g., bright colours and noxious odours). These aversions may be flexible, that is, they can be reinforced or extinguished with experience. As such, we might expect populations to differ in their biases against certain prey characteristics (e.g., colour or pattern) depending on the prey available in the community. Here, we tested whether the jumping spider, Habronattus brunneus(Salticidae), exhibits colour biases against red prey using choice tests with novel (artificially coloured) prey, and then went on to examine how these biases differ across four geographically distinct focal populations. We then conducted preliminary field surveys at each of these four sites to explore differences in habitat type and prey availability. Overall, we found that field‐caught H. brunneusexhibited a bias against the colour red (compared with black) when tested with artificially coloured prey in the laboratory. However, the degree of colour bias varied among our four focal populations (with two populations exhibiting strong biases against red and two showing no colour biases). Preliminary habitat and prey surveys suggest that these populations also differed in both habitat structure (percentage of grass, leaf litter and bare ground) and prey availability across many taxa, suggesting that the spiders from each population may have been exposed to, and had experiences with, different potential prey. We discuss how prey availability may influence colour biases in predators, as well as how predator experience may shape the evolution of prey colour signals.