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The Apalachicola–Chattahoochee–Flint (ACF) basin is arguably the most litigated interstate river system in the eastern United States. Given the complicated demands for water use within this basin, it has been difficult to ascertain if the recent multi-decadal decline in streamflow is a product of human disturbance, changing climate, natural variability, or some combination of the above factors. To overcome these challenges, we examined unimpaired streamflow and precipitation within and adjacent to the ACF basin, upstream of the Apalachicola River at Chattahoochee, and the Florida streamflow station (ARCF), which has historically been identified to be representative of hydrologic variability in the ACF basin. Several of the upstream, unimpaired, streamflow stations selected were identified in rural watersheds where land-cover changes and human disturbance were minimal during the study period. When applying a series of statistical evaluations, ARCF streamflow variability generally reflects the natural variability of the ACF basin. Additionally, unimpaired streamflow variability from the neighboring Choctawhatchee River compared favorably with ARCF variability. The recent multi-decadal decline was consistent in all records, with the 2000s being the most severe in the historic record.