There is currently a need in Spain for monitoring the effects of the environmental flows. The use of eco-hydrological relationships to design both environmental flows regimes and the monitoring programs that track their effects on riverine ecosystems is highly recommended. Fishes are the biological element more sensitive to flow regime changes, thus the relationship between fishes and flow dynamics becomes especially relevant to design environmental flows and the required monitoring programs. This study presents an analysis of the relationships between flow regimes in 33 Spanish rivers, defined using a set of annual, monthly, seasonal, and daily hydrological metrics, including the Indicators of Hydrological Alteration, and fish assemblages, characterized through a combination of taxonomic community composition and ecological traits metrics. A selection of fish and flow metrics that were statistically relevant and non-redundant was conducted, through a Principal Component Analysis and correlation analysis. The potential biological implications of the fish-flow relationships that were found in the statistical analysis were discussed according to the findings in the literature. The results showed that larger rivers tended to have a greater fish richness and a lower fish abundance than smaller rivers. More specifically, rivers that have their annual maximum flows in April were related to a higher fish richness, showing the importance of the timing, and not only the magnitude, of the flow regime. Furthermore, this flow condition may enhance native species richness and control the spread of alien species in rivers downstream from dams. The number of flow reversals was especially suitable when studying fish-hydrological relationships at a national scale, and they were negatively correlated with fish abundance in rivers. In contrast, flow skewness was positively correlated with fish abundance in rivers, and they may promote fish recruitment. Other correlations among some hy, Regardless of their benefits for the society, dams substantially alter the streamflow in rivers. The implementation of environmental flows is a management tool to reduce this alteration. There is currently a need in Spain for monitoring the effects of the environmental flows implemented in rivers. Eco-hydrological relationships provide information about how biological elements are influenced by the streamflow, and they are relevant knowledge to design environmental flows strategies. Fishes are the biological element more sensitive to flow regime changes, and thus the study of fish-flow relationships is particularly relevant in the design of environmental flows. This study presents an analysis of the relationships between flow regimes in 33 Spanish rivers, defined using a set of annual, monthly, seasonal, and daily hydrological metrics, and fish assemblages, characterized through metrics that describe features in rivers such as the number of species, the density of fishes or their habitat preference. A selection of fish and flow metrics that were statistically relevant and non-redundant was conducted through a statistical analysis. Fish-flow relationships were discussed and established based on findings in the literature. The results showed that flow magnitude in April was related to rivers with a higher number of species. Fluctuations in flow magnitude tended to decrease fish density in rivers, while rivers with the variability of high flows tended to have higher values of fish density. Fish metrics related to ecological features showed some relationships with hydrological metrics in the statistical analysis, but they were not reliable enough, as the findings were not supported by the literature.