Regulators have realized the need for ecosystem-based water resources management that goes beyond the typical focuses on flood protection and water quality control. Understanding and incorporating the connections between hydrology and ecology is important in furthering holistic watershed-scale resources protection. This paper describes the characterization of in-channel habitat mosaics, which result from the interactions between flow and physical channel features, in providing flow-specific areas of refuge and connectivity through a second-order agricultural stream. Fish communities are used to compare the dynamic nature of habitat mosaics at different reach locations. Analyses of the communities illustrate the role of mosaic characterization in understanding the importance of hydrologic connectivity in watershed management and stream restoration. The results of this study promote understanding of the links between physical and ecological processes, as well as the effects of source areas, habitat suitability, and mosaic dynamics on fish communities.