Indices of hypoxia and hyperthermia tolerance for Missouri fish assemblages were based on laboratory measurements of lethal dissolved oxygen concentrations and temperatures, combined with field measures of the relative abundances of tolerant and sensitive species. Fish assemblages and extreme physicochemical conditions were monitored over 3–4 years at 18 sites on headwater streams in the Prairie, Ozark Border, and Ozark regions of Missouri. Oxygen minima ranged from 0.8 to 6.0 mg/L, and temperature maxima ranged from 19.6 to 30.7°C; oxygen minima at study sites were not correlated with temperature maxima. Hypoxia tolerances of fish assemblages were strongly correlated with minimum stream oxygen concentrations and varied concordantly with regional, longitudinal, and temporal gradients in stream oxygen minima. Hyperthermia tolerances of fish assemblages were not correlated with maximum stream temperatures, nor were regional, longitudinal, or temporal differences in hyperthermia tolerances concordant with variation in temperature maxima. Axis scores from a detrended correspondence analysis of species frequencies were strongly correlated with dissolved oxygen minima for all 18 sites, but axis scores correlated with temperature maxima only at the four well-oxygenated sites. Low dissolved oxygen levels had a substantial effect on the composition of fish assemblages at most sites, but maximum temperatures influenced assemblages only at the few sites without severe levels of hypoxia.