The existing research evidence indicates that gender-specific travel behavior is poorly understood, and there is still a lack of knowledge about to what extent cycling route choice behavior of women varies significantly from that of men. This study is the first to reveal gender aspects in route choice of cyclists in the German context, based on revealed preference data. We employ a model which allows for a direct comparison of all preferences in terms of gender, and apply it on a well-balanced sample of bicycle route data collected through GPS tracking in a country with a gender-neutral cycling scenario. We include attributes such as surface type, elevation, bicycle infrastructure, or road type. We find, for example, that the sheer existence of bicycle infrastructure is more important to women than to men, but the type and quality of bicycle infrastructure has a higher importance for male cyclists. The route choice model indicates a high value of goodness-of-fit measure. Future research will encompass analysis on much bigger datasets and application of modelling approaches capturing unobserved heterogeneity.