Road networks threaten biodiversity and particularly herpetofauna, including common snapping turtles (Chelydra serpentina), which have an especially slow life history that prevents rapid recovery of populations subjected to road mortality. Cootes Drive is a 2.5-km 4-lane highway that bisects wetland habitat used for nesting and overwintering by snapping turtles. We hypothesized that turtle mortality from collisions with vehicles on Cootes Drive has caused a male bias and a decline in the population as turtles attempt to access habitat on both sides of the road. Capture–mark–recapture studies confirmed a dramatic decline in the turtle population from 941 individuals in 1985 to 177 individuals in 2002, a loss of 764 individuals in only 17 yrs. Using the same data, we also determined that the population has been significantly male-biased since 1985. Using 2009–2016 road mortality data obtained from the Dundas Turtle Watch (a citizen-science program), we completed a population viability analysis using the 2002 population size estimate to isolate the impact of road mortality. We found that this population is at risk of extirpation due to road mortality. The population range overlapped with the Cootes Drive and 7 of the 10 tracked turtles had individual home ranges that overlapped with the road. Our findings support the hypothesis that road mortality has contributed to the dramatic decline in the snapping turtle population in Cootes Paradise Marsh. This population is in jeopardy of extirpation; therefore, exclusion fencing must be installed for an extended distance along both sides of surrounding roads to prevent turtles from crossing the road and to promote their use of existing aquatic culverts.