Providing a sufficiently appropriate route environment is crucial to ensuring fair andsafe biking, thus encouraging cycling as a sustainable mode of transport. At the same time,better understanding of cyclists’ preferences regarding the features of their routes and theirinfrastructure requirements is fundamental to evaluating improvement of the current infrastructureor the development of new infrastructure. The present study has two objectives. The first is toinvestigate cyclists’ route preferences by means of a choice experiment based on a stated preferencesurvey. Subsequently, the second objective is to compare cyclist preferences in two countries withdifferent cycling characteristics (both in infrastructure as well as cyclists’ behavior). For this purpose,a graphical online stated preferences survey was conducted in Greece and Germany. Within theframework of statistical analyses, multinomial mixed logit discrete choice models were developedthat allow us to quantify the trade-offs of interest, while distinguishing between the preferences ofdifferent user groups. In addition, user requirements in Greece, as a country with a low cycling shareand very little dedicated bike infrastructure, were compared to the requirements in Germany, wherecycling is popular and the infrastructure is well developed. The results over the whole sample indicatethat subgroups value infrastructure differently according to their specific needs. When looking atcountry specifics, users from Greece are significantly more willing to accept longer travel times inreturn for higher-quality facilities. The utility of low speed limits in mixed traffic is also different. InGermany, low speed limits offset the disturbance caused by motorized traffic, but in Greece they donot. Consequently, the results help to asses which types of infrastructure are most sustainable from auser perspective and help to set priorities when the aim is to adapt the road infrastructure efficientlyin a stable strategy.