To determine the prevalence of mycotoxins in streams, 116 water samples from 32 streams and three wastewater treatment plant effluents were collected in 2010 providing the broadest investigation on the spatial and temporal occurrence of mycotoxins in streams conducted in the United States to date. Out of the 33 target mycotoxins measured, nine were detected at least once during this study. The detections of mycotoxins were nearly ubiquitous during this study even though the basin size spanned four orders of magnitude. At least one mycotoxin was detected in 94% of the 116 samples collected. Deoxynivalenol was the most frequently detected mycotoxin (77%), followed by nivalenol (59%), beauvericin (43%), zearalenone (26%), β-zearalenol (20%), 3-acetyl-deoxynivalenol (16%), α-zearalenol (10%), diacetoxyscirpenol (5%), and verrucarin A (1%). In addition, one or more of the three known estrogenic compounds (i.e. zearalenone, α-zearalenol, and β-zearalenol) were detected in 43% of the samples, with maximum concentrations substantially higher than observed in previous research. While concentrations were generally low (i.e. < 50 ng/L) during this study, concentrations exceeding 1000 ng/L were measured during spring snowmelt conditions in agricultural settings and in wastewater treatment plant effluent. Results of this study suggest that both diffuse (e.g. release from infected plants and manure applications from exposed livestock) and point (e.g. wastewater treatment plants and food processing plants) sources are important environmental pathways for mycotoxin transport to streams. The ecotoxicological impacts from the long-term, low-level exposures to mycotoxins alone or in combination with complex chemical mixtures are unknown