Effluent-fed streams, which receive inputs from wastewater treatment plants, are becoming increasingly common across the globe as urbanization intensifies. In semi-arid and arid regions, where many natural streams have dried up due to over extraction of water, many streams rely completely on treated effluent to sustain baseflow during dry seasons. These systems are often thought of as ‘second-class’ or highly disturbed stream ecosystems, but they have the potential to serve as refuges for native aquatic biota if water quality is high, especially in areas where few natural habitats remain. In this study, we investigated seasonal and longitudinal water quality dynamics at multiple sites across six reaches of three effluent-dependent rivers in Arizona (USA) with the objective (1) to quantify changes in effluent water quality due to distance traveled and season/climate and (2) to qualify whether water quality conditions in these systems are sufficient to support native aquatic species. Study reaches ranged in length from 3 to 31 km and in geographic setting from low desert to montane conifer forest. We observed the lowest water quality conditions (e.g., elevated temperature and low dissolved oxygen) during the summer in low desert reaches, and significantly greater natural remediation of water quality in longer vs. shorter reaches for several factors, including temperature, dissolved oxygen and ammonia. Nearly all sites met or exceeded water quality conditions needed to support robust assemblages of native species across multiple seasons. However, our results also indicated that temperature (max 34.2 °C), oxygen levels (min 2.7 mg/L) and ammonia concentrations (max 5.36 mg/L N) may occasionally be stressful for sensitive taxa at sites closest to effluent outfalls. Water quality conditions may be a concern during the summer. Overall, effluent-dependent streams have the capacity to serve as refuges for native biota in Arizona, and they may become the only aquatic habitat available in many urbanizing arid and semi-arid regions.