Understanding the future impacts of climate and land use change are critical for long-term biodiversity conservation. We developed and compared two indices to assess the vulnerability of stream fish in Missouri, USA based on species environmental tolerances, rarity, range size, dispersal ability and on the average connectivity of the streams occupied by each species. These two indices differed in how environmental tolerance was classified (i.e., vulnerability to habitat alteration, changes in stream temperature, and changes to flow regimes). Environmental tolerance was classified based on measured species responses to habitat alteration, and extremes in stream temperatures and flow conditions for one index, while environmental tolerance for the second index was based on species’ traits. The indices were compared to determine if vulnerability scores differed by index or state listing status. We also evaluated the spatial distribution of species classified as vulnerable to habitat alteration, changes in stream temperature, and change in flow regimes. Vulnerability scores were calculated for all 133 species with the trait association index, while only 101 species were evaluated using the species response index, because 32 species lacked data to analyze for a response. Scores from the trait association index were greater than the species response index. This is likely due to the species response index's inability to evaluate many rare species, which generally had high vulnerability scores for the trait association index. The indices were consistent in classifying vulnerability to habitat alteration, but varied in their classification of vulnerability due to increases in stream temperature and alterations to flow regimes, likely because extremes in current climate may not fully capture future conditions and their influence on stream fish communities. Both indices showed higher mean vulnerability scores for listed species than unlisted species, which provided a coarse measure of validation. Our indices classified species identified as being in need of conservation by the state of Missouri as highly vulnerable. The distribution of vulnerable species in Missouri showed consistent patterns between indices, with the more forest-dominated, groundwater fed streams in the Ozark subregion generally having higher numbers and proportions of vulnerable species per site than subregions that were agriculturally dominated with more overland flow. These results suggest that both indices will identify similar habitats as conservation action targets despite discrepancies in the classification of vulnerable species. Our vulnerability assessment provides a framework that can be refined and used in other regions.