Fishermen often anecdotally report an unexpected increase of fish caught in small tributary streams during floods, presumably due to refuge-seeking behavior from the main stem. From a population perspective, this implies the significance of refuge habitats and connectivity for population viability against natural disturbances. Despite the plausibility, however, surprisingly few studies have examined the tributary refuge hypothesis, mainly due to the difficulty in field survey during floods. Here, we made use of a large-scale controlled flood to assess whether fishes move into tributaries during flooding in the main stem. A planned water release from the Satsunai River Dam located on Hokkaido Island in Japan rapidly increased the main stem discharge by more than 20-fold. Before, during, and after flooding censuses in four tributaries provided evidence of the refuge-seeking behavior of fishes from the main stem. For example, more than 10 Dolly Varden char, a salmonid fish, were caught in a tributary during the flood, even though almost no individuals were captured before or after the flood. The fish responded immediately to the flooding, suggesting the need for studies during disturbances. In addition, the likelihood of refuge movements varied among tributaries, suggesting the importance of local environmental differences between tributary and the main stem habitats. This is the first study to experimentally confirm the tributary refuge hypothesis, and underscores the roles of habitat diversity and connectivity during disturbances, even though some habitats are not used during normal conditions.