Traditional analysis in population genetics evaluates differences among groups of individuals and, in some cases, considers the effects of distance or potential barriers to gene flow. Genetic variation of organisms in complex landscapes, seascapes, or riverine systems, however, may be shaped by many forces. Recent research has linked habitat heterogeneity and landscape or seascape configuration to genetic structure by integrating methods from landscape ecology, population genetics, and spatial statistics in approaches known as landscape or seascape genetics. However, functional differences between terrestrial or seascapes systems in comparison to riverscape topography (i.e., movement pathways for aquatic obligate species are constrained to river channels) make translating these approaches into freshwater analyses problematic. Studies that may be described as riverscape genetics (RG) have linked temperature, stream gradient, and confluences to genetic variability. Lack of consistency in methodology, however, has made comparisons across species and scales difficult. We provide a perspective on how RG could be used to provide a more comprehensive conceptual and applied understanding of connectivity and dispersal in freshwater systems. We describe four thematic areas of study representing current and future research opportunities and describe a basic workflow for conducting RG analysis. Although numerous methodological challenges remain, a RG approach can enhance our understanding of habitat heterogeneity in shaping gene flow and spatial genetic structure. These characteristics of populations are critical components for interpreting demographic and evolutionary consequences of habitat loss and fragmentation.