Public concern regarding per- and polyfluoroalkyl substances (PFAS) has grown substantially in recent years. In addition, research has documented multiple potential agriculture-related release pathways for PFAS (e.g., biosolids and livestock manure). Nevertheless, little research on the environmental prevalence of PFAS has been conducted in agricultural regions of the United States. To fill this gap, we conducted the first statewide spatiotemporal assessment of PFAS in Iowa streams across a region of intense agricultural activity. At least one PFAS was detected at 19 of the 60 stream sites sampled (32%) with 10 different PFAS detected statewide. The number of PFAS detected in the stream samples ranged from one to nine. While PFAS were detected in agricultural streams, sites with the most PFAS detected and in the highest concentration were small, effluent-affected streams where wastewater treatment plant discharge is driving stream PFAS concentrations. No individual PFAS had an exposure:activity ratio (EAR) of >1.0 (exposure concentration shown to trigger observed adverse biological activity). Five stream locations, however, had at least one EAR of >0.001, a precautionary effect screening threshold. Additional targeted temporal sampling would be beneficial to specifically capture potential agricultural source applications and corresponding runoff conditions to fully characterize the prevalence of PFAS in such agricultural systems.