Surface water quality impairment in agricultural watersheds is a major environmental concern in the United States. To assess seasonal and spatial variability of surface water quality and identify factors associated with surface water quality variability, we monitored surface water quality at seven locations in Bayou Plaquemine Brule Watershed in Louisiana twice monthly from March of 2002 to February of 2008 and performed multivariate analyses of the dataset. Using the Soil and Water Assessment Tool (SWAT) model, we identified critical areas of nonpoint source pollution in the watershed. While temperature, turbidity, dissolved oxygen (DO), conductivity and pH were determined in the field using YSI Sonde (YSI Incorporated, Yellow Springs, Ohio), surface water samples were analyzed for total nitrogen (TN), total phosphorus (TP), nitrate/nitrite-N (NO 3 /NO 2 -N), soluble reactive phosphate (SRP), total suspended solids (TSS), and five-day biological oxygen demand (BOD 5 ) in laboratory. The monthly water quality sampling included a regular sampling and an after-rain-event sampling. Average DO for the summer months, March through November, was 4.91 ± 0.08 mg L -1 (4.91 ± 0.08 ppm), while average DO for the winter months, December through February, was 8.32 ± 0.12 mg L -1 (8.32 ± 0.12 ppm). Dissolved oxygen was negatively correlated with TN (r = -0.22, p ≤ 0.001), SRP (r = -0.17, p ≤ 0.001), TP (r = -0.17, p ≤ 0.001), BOD 5 (r = -0.25, p ≤ 0.001), and surface water temperature (r = -0.70, p ≤ 0.001). Turbidity was strongly correlated with TSS (r = 0.59, p ≤ 0.001), suggesting that most turbidity in the water body comes from suspended solids. Similarly BOD 5 was significantly positively correlated with TN (r = 0.43, p ≤ 0.001), NO 3 /NO 2 -N (r = 0.26, p ≤ 0.001), TP (r = 0.25, p ≤ 0.001), and SRP (r = 0.18, p ≤ 0.001). Results of factor analyses showed sediment, phosphorus (P), nitrogen (N), surface water temperature, dissolved solids, and acidity/alkalinity as the most important factors associated with surface water quality variability in this watershed. Although relatively higher concentrations of sediments, TP, and TN were observed in the upper reaches of the watershed based on water quality monitoring, the SWAT simulation results showed the critical nonpoint source pollution areas of sediment, P, and N in the lower reaches of the watershed. Lower reaches of the watershed have mainly rice and crawfish production, while the upper reaches include primarily sugarcane, pasturelands, and soybean production. Information on seasonal variability of surface water quality, factors associated with surface water quality variability, and the critical areas for nonpoint source pollution will be valuable inputs for developing a watershed management plan for effective nonpoint source pollution control in an agricultural watershed.