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Urbanization is a significant driver of the global biodiversity crisis. Turtles are particularly impacted by urbanization because of the vulnerability of riparian habitats to habitat loss and road mortality. Behaviour plays a crucial role in determining the success of urban animals. Behavioural responses to urbanization, however, are rarely studied in turtles even though many turtles are at‐risk and sometimes live in urban areas. Therefore, we evaluated behavioural changes in painted turtles (Chrysemys picta) living in wetlands surrounded by a gradient of urbanization. We tested the consistency of painted turtle behaviour in the laboratory and examined the behaviour of painted turtles from 24 wetland sites across an urbanization gradient in Ottawa, Ontario, Canada. We assessed: (i) aggression by measuring the number of active defensive behaviours the turtles performed in response to handling, (ii) boldness by measuring the amount of time the turtles took to emerge from their shells and move from their initial locations in a circular arena and (iii) activity by measuring the amount of time the turtles spent moving in the same circular arena. We found that all behaviours were consistent in the laboratory. We also found that as the level of urbanization increased, turtles were more aggressive and bolder. Urbanization affects painted turtle behaviour, but further research is required to understand the mechanisms responsible and the conservation implications. [ABSTRACT FROM AUTHOR]