Headwater streams are the smallest, yet most abundant reaches in any river network and comprise most of the stream length; as a result, they drain the vast majority of terrestrial landscapes across the globe. These small but numerous ecosystems are essential components in the structure and function of larger streams and rivers, wetlands, and receiving lentic water bodies including lakes as well as marine systems. Despite the prominence of headwaters, their role as permanent or temporary fish habitat remains understudied, including the degree to which they’re used by freshwater fishes, one of the most threatened taxa groups globally. While many fishes are described in the literature as accessing headwaters for portions of their life cycles, a large-scale assessment documenting species presences in headwaters has not occurred. To meet this need, we characterize the extent to which freshwater fishes occur in headwater streams across the United States. First, using an existing database characterizing presence of species from nearly 60,000 headwater stream reaches, we document the frequency with which species occur in these habitats, and we include results summarized for endemic, rare, and threatened species. Additionally, we summarize and map the degree to which headwater habitats supporting these species are currently protected based on the Protected Areas Database of the United States, thus highlighting the vast number of species relying on headwater habitats and the mismatch in the protection of these catchments. Finally, we summarize literature describing assumed headwater dependency of hundreds of fishes identified from our analyses. These results help to amplify awareness of the elevated vulnerability of many freshwater fishes stemming from recent legislative changes in the Clean Water Act, which removes many headwaters from federal protection and from changes in how the Endangered Species Act treats protection of habitat supporting imperiled species. Collectively, our results show the overall importance of headwater habitats to freshwater fishes and underscores the need to conserve these critical habitats for our most vulnerable species.