The quality of input data and the process of watershed delineation can affect the accuracy of runoff predictions in watershed modeling. The Upper Mississippi River Basin was selected to evaluate the effects of subbasin and/or hydrologic response unit (HRU) delineations and the density of climate dataset on the simulated streamflow and water balance components using the Hydrologic and Water Quality System (HAWQS) platform. Five scenarios were examined with the same parameter set, including 8- and 12-digit hydrologic unit codes, two levels of HRU thresholds and two climate data densities. Results showed that statistic evaluations of monthly streamflow from 1983 to 2005 were satisfactory at some gauge sites but were relatively worse at others when shifting from 8-digit to 12-digit subbasins, revealing that the hydrologic response to delineation schemes can vary across a large basin. Average channel slope and drainage density increased significantly from 8-digit to 12-digit subbasins. This resulted in higher lateral flow and groundwater flow estimates, especially for the lateral flow. Moreover, a finer HRU delineation tends to generate more runoff because it captures a refined level of watershed spatial variability. The analysis of climate datasets revealed that denser climate data produced higher predicted runoff, especially for summer months.