1. Using data from 270 minimally disturbed river sites distributed across France, we examined relationships between fish assemblages and four sets of explanatory variables representing climate, position in the river network, geomorphology and flow regime (temporal flow variation).
2. Flow regimes, represented by 157 hydrological indices, were derived from 763 gauging stations representing unmodified flows. Principal components analysis was used to reduce the 157 indices to nine non-redundant hydrological variables, and hydrological regionalisation was then used to transfer the hydrological variables to the sampling sites.
3. Climate, position in the river network and geomorphology, collectively referred to as ‘habitat’ factors, and were represented by the variables mean January and July temperature, distance from source, mean flow and altitude and width, depth, slope, sediment size and % lotic habitat.
4. Partial constrained ordination (pCCA) showed that hydrological variables individually explained 9.7% of the variability of fish assemblages, however the variation explained by the hydrological variables decreased to only 3.4% when the variation explained by the habitat variables was removed.
5. Our study showed that co-variation between hydrological and habitat variables need to be accounted for to avoid overestimating the importance of biological–hydrological relationships. Failing to do so may have important implications for the development of environmental flow prescriptions, as apparent biological responses to altered flow regimes are likely to be confounded by variation associated with habitat variability.