The global expansion and increased intensity of the human footprint causes modification of natural freshwater systems, particularly in densely populated areas. This poses a challenge for society where the water systems that humans rely on for domestic, industrial, and agricultural water are also the most physically and chemically disturbed. There is an urgent need to study hydrologic processes in surface water systems that flow through human-altered landscapes to better manage freshwater resources. Stable water isotopes (δ18O and δ2H) can be used as natural tracers to study the hydrologic patterns of rivers as they flow through these agricultural and urban landscapes. In this work, we investigate the seasonal and spatial hydrologic patterns of the Scioto River in Ohio, USA by analyzing stable water isotopes. Samples were collected over the course of one year (2021) from five locations that transition from intense agriculture to the metropolitan area of Columbus, Ohio. Results show that the Scioto River isotopic composition reflects a dampened seasonal precipitation pattern, where δ18O and δ2H are enriched in summer and depleted in winter. Scioto River transect patterns show that there was greater variability in stable isotopic composition in the upstream, agricultural reaches of the river. As the river flowed through the metropolitan Columbus area two “river-like” reservoirs generated isotopic homogenization along the Scioto without inducing isotopic enrichment, which is commonly observed from evaporation in river-reservoir systems. The Scioto River was put in context with other major Ohio river systems, to show the variability across the state. Isotopic composition differences among and within river systems across the state were induced by a combination of different environmental factors, like precipitation and river baseflow contribution. They were further affected by humans through land use modification and the constructions of dams and reservoirs. Overall, this study underlines the influence that urbanization and intensive agriculture have on rivers. In the context of the current hydrologic changes induced by climate change, this work emphasizes the importance of studying urban river hydrology in the context of joint climatic and human influences.