<em>Abstract</em>.—Our capabilities to address pertinent questions in stream fish ecology, such as population connectivity, biotic homogenization, species invasions, introgression, and effects of habitat alterations on population structure and demography, have been significantly expanded by the development of molecular genetic approaches. A broad spectrum of molecular markers can now be tailored to address specific questions while newer statistical approaches accommodate larger data sets and permit the test of alternative hypotheses. Furthermore, molecular approaches facilitate the evaluation of ecological processes across both spatial and temporal scales, which are often mutually exclusive parameters. Population expansions, declines, and movements can be examined from recent to deep history and scaled from local to continental drainages. The intrinsic properties of stream ecosystems also make them particularly amenable to molecular approaches. The hierarchical order reflected in streams is directly translatable into an expanding spatial scale, from restricted headwaters through entire basins. Additionally, stream habitats are generally linear, and consequently, fish populations are often distributed sequentially, with interactions constrained to neighboring populations. Finally, streams tend to develop vicariant barriers over time, thus isolating populations and promoting local adaptation, a process easily deciphered using molecular markers. The latter have also contributed to the resolution of conservation issues and guided appropriate adaptive management of stream fauna. Molecular approaches in stream fish ecology are far too diverse to be comprehensively reviewed herein. Instead, we illustrate their facility by emphasizing three case studies demonstrating their broad utility: (1) a range-wide analysis of mitochondrial DNA diversity in flannelmouth sucker <em>Catostomus latipinnis</em>, pointing to a population bottleneck likely induced by severe post-Pleistocene drought in the Colorado River basin; (2) single nucleotide polymorphism screening to evaluate hybridization and introgression among native flannelmouth sucker, bluehead sucker <em>C. discobolus </em>(also known as <em>Pantosteus discobolus</em>), and the introduced white sucker <em>C. commersonii </em>in the upper Colorado River basin; and (3) microsatellite DNA analysis to evaluate gene flow and contemporary relationships in the Grand Canyon among populations of an endangered cyprinid fish (the humpback chub <em>Gila cypha</em>). In an appendix, we outline several recent molecular approaches that have expanded our opportunities to study stream fish ecology. We review relevant literature by emphasizing new statistical approaches and potential pitfalls of marker selection and data, rather than by delving into abstruse technical details regarding protocol development.