Objective. Females are substantially less likely than males to cycle for transport in countries with low bicycle transport mode share. We investigated whether female commuter cyclists were more likely to use bicycle routes that provide separation from motor vehicle traffic. Methods. Census of cyclists observed at 15 locations (including off-road bicycle paths, on-road lanes and roads with no bicycle facilities) within a 7.4 km radius of the central business district (CBD) of Melbourne, Australia, during peak commuting times in February 2004. Results. 6589 cyclists were observed, comprising 5229 males (79.4%) and 1360 females (20.6%). After adjustment for distance of the bicycle facility from the CBD, females showed a preference for using off-road paths rather than roads with no bicycle facilities (odds ratio [OR] = 1.43, 95% confidence interval [CI]: 1.12, 1.83), or roads with on-road bicycle lanes (OR = 1.34, 95% CI: 1.03, 1.75). Conclusions. Consistent with gender differences in risk aversion, female commuter cyclists preferred to use routes with maximum separation from motorized traffic. Improved cycling infrastructure in the form of bicycle paths and lanes that provide a high degree of separation from motor traffic is likely to be important for increasing transportation cycling amongst under-represented population groups such as women.