Road mortality is thought to be a leading cause of turtle population decline. However, empirical evidence of the direct negative effects of road mortality on turtle population abundance is lacking. The purpose of this study was to provide a strong test of the prediction that roads reduce turtle population abundance. While controlling for potentially confounding variables, we compared relative abundance of painted turtles (Chrysemys picta) in 20 ponds in Eastern Ontario, 10 as close as possible to high traffic roads (Road sites) and 10 as far as possible from any major roads (No Road sites). There was no significant effect of roads on painted turtle relative abundance. Furthermore, our data do not support other predictions of the road mortality hypothesis; we observed neither a higher relative frequency of males to females at Road sites than at No Road sites, nor a lower average body size of turtles at Road than at No Road sites. We speculate that, although roads can cause substantial adult mortality in turtles, other factors, such as release from predation on adults and/or nests close to roads counter the negative effect of road mortality in some populations. We suggest that road mitigation for painted turtles can be limited to locations where turtles are forced to migrate across high traffic roads due, for example, to destruction of local nesting habitat or seasonal drying of ponds. This conclusion should not be extrapolated to other species of turtles, where road mortality could have a larger population-level effect than on painted turtles.