The three grayling Thymallus thymallus age‐classes had strong preferences for their local habitat in the Ain river. All age‐classes preferred high water velocities between 70 and 110 cm s‐1, confirming that grayling is a typical rheophilic species. An important intraspecific segregation regarding depth occurred, as large individuals preferred deeper water than small ones (optimal ranges=50–60, 80–120 and 100–140 cm for 0+, 1+ and adults respectively). The three age‐classes had similar preferences for small substratum articles, with optimal values between 0·5 and 16·0 mm. Preference curves were generally comparable between the two sites, despite important differences in habitat availability. It is emphasized that grayling needs various habitat conditions to achieve its entire life cycle. The increasing scarcity of this species underlines the urgency of protecting lotic habitat integrity and diversity.