Turtles are particularly susceptible to the negative impacts of urbanization due to low mobility and a life history strategy emphasizing long generation times and high adult survival. In addition to declines directly through habitat loss, urbanization has been hypothesized to limit populations of aquatic turtles through changes in population structure, as adult females are disproportionally killed on and near roads, leading to male-biased populations, which can lead to population declines or local extirpations. The purpose of this study was to better understand how urbanization impacts the sex ratios of painted turtles (Chrysemys picta) in an urban ecosystem, as empirical results linking male-biased turtle populations to roads and urbanization are mixed. Using eight years of trapping data from a long-term monitoring program in a suburb of Chicago, IL, USA, we report one of the most male-biased populations (x = 75% male) of turtles in the USA, consistent with prevailing road mortality hypotheses. However, we found no evidence that male-biased populations were related to road density or the amount of protected area around a sampling location and found that impervious surface (a metric of urbanization) was weakly related to less male-biased populations. Our results highlight the importance of replicating ecological studies across space and time and the difficulty in assessing population structure in aquatic turtles. We suggest that active conservation measures may be warranted for the continued persistence of urban turtle populations.Diversity 2019, 11, 72 2 of 13 and degradation [10,13], road mortality [11,[14][15][16], and increased depredation rates by subsidized predators [17][18][19][20]. Understanding how turtles respond to urbanization is imperative in a world with a rapidly increasing urban footprint [4,21] and rapidly shrinking turtle populations [22,23].Urbanization has been hypothesized to further limit populations of aquatic turtles through changes in population structure, as adult females (relative to males and juveniles) are disproportionally killed on and near roads [24][25][26][27][28], and Gibbs and Steen [27] show that turtle populations in the US have become more male-biased over time, coinciding with the proliferation of roads. This problem is particularly insidious as turtle populations can become functionally extinct well before the last turtle in a population actually dies [22,29]. However, empirical results of urbanization impacting the sex ratios of turtle populations are mixed and limited to two species with a limited geographic scope [25,[30][31][32], and a series of recent papers has failed to find an association between roads and male-biased populations [33][34][35][36]. Notably, Bowne et al. [36] found the opposite relationship, and hypothesized that that urbanization could be resulting in warmer nesting locations, leading to more females being produced via environmental sex determination [37] (but see Lambert and Steen [38]).In this study, we attempt to better understand how urbanizat...