Despite the importance of the Missouri River for navigation, recreation, habitat, hydroelectric power, and agriculture, relatively little is known about the basic hydroclimatology of the Missouri River basin (MRB). This is of particular concern given the droughts and floods that have occurred over the past several decades and the potential future exacerbation of these extremes by climate change. Here, observed and modeled hydroclimatic data and estimated natural flow records in the MRB are used to 1) assess the major source regions of MRB flow, 2) describe the climatic controls on streamflow in the upper and lower basins , and 3) investigate trends over the instrumental period. Analyses indicate that 72% of MRB runoff is generated by the headwaters in the upper basin and by the lowest portion of the basin near the mouth. Spring precipitation and temperature and winter precipitation impacted by changes in zonal versus meridional flow from the Pacific Ocean play key roles in surface water supply variability in the upper basin. Lower basin flow is significantly correlated with precipitation in late spring and early summer, indicative of Atlantic-influenced circulation variability affecting the flow of moisture from the Gulf of Mexico. Although increases in precipitation in the lower basin are currently overriding the effects of warming temperatures on total MRB flow, the upper basin’s long-term trend toward decreasing flows, reduction in snow versus rain fraction, and warming spring temperatures suggest that the upper basin may less often provide important flow supplements to the lower basin in the future.