Although a number of studies have investigated pollutant transport patterns in urban watersheds, these studies have focused primarily on the upland landscape as the point of interest (i.e., prior to stormwater entering an open stream channel). However, it is likely that in-stream processes will influence pollutant transport when the system is viewed at a larger scale. One initial investigation that can be performed to characterize transport dynamics in urban runoff is determining a pollutant’s temporal distribution. By borrowing from urban stormwater literature, the propensity of a pollutant within a system to be more heavily transported in the initial portion of the storm can be quantified (i.e., the “first flush”). Although uncommon for use in stream science, this methodology allows direct comparison of results to previous studies on smaller urban upland catchments. Multiple methods have been proposed to investigate the first flush effect, two of which are applied in this study to two streams in Knoxville, TN, USA. The strength of the first flush was generally corroborated by the two unique methods, a new finding that allows a more robust determination of first flush presence for a given pollutant. Further, an “end flush” was observed and quantified for nutrients and microbes in one stream, a novel outcome that shows how the newer methodology that was employed can provide greater insight into transport processes and pollutant sources. Explanatory variables for changes in each pollutant’s inter-event first flush strength differed, but notable relationships included the influence of flow rate on microbes and influence of rainfall on Cu²⁺. The results appear to support the hypothesis that in-stream processes, such as resuspension, may influence pollutant transport in urban watersheds, pointing toward the need to consider in-stream processes in models developed to predict urban watershed pollutant export.