The purpose of this study is to evaluate the uncertainty attributable to inadequate temporal sampling of stormwater discharge and water quality, and understand its implications for meeting monitoring objectives relevant to municipal separate storm sewer systems (MS4s). A methodology is presented to evaluate uncertainty attributable to inadequate temporal sampling of continuous stormflow and water quality, and a case study demonstrates the application of the methodology to six small urban watersheds (0.8–6.8km2 0.8 – 6.8    km 2 ) and six large rural watersheds (30–16,192km2 30 – 16,192    km 2 ) in Virginia. Results indicate the necessity of high-frequency continuous monitoring for accurately capturing multiple monitoring objectives, including illicit discharges, acute toxicity events, and stormflow pollutant concentrations and loads, as compared to traditional methods of sampling. For example, 1-h sampling in small urban watersheds and daily sampling in large rural watersheds would introduce uncertainty in capturing pollutant loads of 3–46% and 10–28%, respectively. Overall, the outcomes from this study highlight how MS4s can leverage continuous monitoring to meet multiple objectives under current and future regulatory environments.