Glyphosate and atrazine are the most intensively used herbicides in the United States. Although there is abundant spatial and temporal information on atrazine occurrence at regional scales, there are far fewer data for glyphosate, and studies that compare the two herbicides are rare. We investigated temporal patterns in glyphosate and atrazine concentrations measured weekly during the 2013 growing season in 100 small streams in the Midwestern United States. Glyphosate was detected in 44% of samples (method reporting level 0.2 μg/L); atrazine was detected above a threshold of 0.2 μg/L in 54% of samples. Glyphosate was detected more frequently in 12 urban streams than in 88 agricultural streams, and at concentrations similar to those in streams with high agricultural land use (> 40% row crop) in the watershed. In contrast, atrazine was detected more frequently and at higher concentrations in agricultural streams than in urban streams. The maximum concentration of glyphosate measured at most urban sites exceeded the maximum atrazine concentration, whereas at agricultural sites the reverse was true. Measurement at a 2-day interval at 8 sites in northern Missouri revealed that transport of both herbicide compounds appeared to be controlled by spring flush, that peak concentration duration was brief, but that peaks in atrazine concentrations were of longer duration than those of glyphosate. The 2-day sampling also indicated that weekly sampling is unlikely to capture peak concentrations of glyphosate and atrazine.