We studied the multiscale (sites, river reaches and rivers) and short-term temporal (monthly) variability in a freshwater fish assemblage. We found that small-scale spatial variation and short-term temporal variability significantly influenced fish community structure in the Macquarie and Namoi Rivers. However, larger scale spatial differences between rivers were the largest source of variation in the data. The interaction between temporal change and spatial variation in fish community structure, whilst statistically significant, was smaller than the variation between rivers. This suggests that although the fish communities within each river changed between sampling occasions, the underlying differences between rivers were maintained. In contrast, the strongest interaction between temporal and spatial effects occurred at the smallest spatial scale, at the level of individual sites. This means whilst the composition of the fish assemblage at a given site may fluctuate, the magnitude of these changes is unlikely to affect larger scale differences between reaches within rivers or between rivers. These results suggest that sampling at any time within a single season will be sufficient to show spatial differences that occur over large spatial scales, such as comparisons between rivers or between biogeographical regions.