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The purpose of the total maximum daily load (TMDL) process is to protect and maintain designated beneficial uses of surface water (e.g., drinking water, livestock watering, irrigation, or fishing). This process require monitoring and assessment of quality of surface water bodies. The specific objectives of this study were to (1) determine the natural variability in the concentration of a non-point source pollutant (e.g., nitrate) and (2) to estimate minimum sample size needed to adequately describe TMDL process. Water samples were collected from nine sites in the Miracle Mile, North Platte River, Wyoming on six sampling dates. The temperature, dissolved oxygen (DO) and pH were measured in the field prior to the sampling. Samples were taken to the Water Quality laboratory and analyzed for nitrate with Ion Chromatography (IC). The temperature of the water was between 13 and 16.8 O C and pH was from 7.6 to 8.9. The DO ranged between 59 and 137% saturation. These water quality parameters increased with distance. Nitrate-nitrogen concentrations ranged from 0.15 to 14.16 mg/L and yielded no consistent trends. Results in this study suggest that to adequately characterize a non-point source pollutant such as nitrate in a natural fluvial ecosystem require large sample sizes.