Land cover change plays a critical role in influencing hydrological responses. Change in land cover has impacted runoff across basins with substantial human interference; however, the impacts in basins with minimal human interference have been studied less. In this study, we investigated the impacts of directional land cover changes (forest to/from combined grassland and shrubland) in runoff coefficient (RC; ratio of runoff to precipitation) and runoff volume across 603 low human interference reference basins in the conterminous United States (CONUS). The results indicate basins with significant (p<0.05) increasing trends in runoff and RC were across the northeast and northwest regions of CONUS, and basins with decreasing trends were in the southern CONUS region. A unit percent increase in basin area from grassland and shrubland to forest was associated with a ∼4% decrease in RC across basins with decreasing RC trends. Similarly, a unit percent increase in basin area from forest to a combined grassland and shrubland was associated with a ∼1% increase in RC across increasing RC trend basins. Runoff volume was decreased (increased) by ∼25 × 10⁶ m³ yr⁻¹ (∼9 × 10⁶ m³ yr⁻¹) across basins with decreasing (increasing) trends in runoff and RC. When relating runoff volume with the area of directional land cover changes, each 1 km² increase in area from grassland and shrubland to forest resulted in a decrease of ∼530,000 m³ runoff volume across basins with decreasing trends. In contrast, each 1 km² increase in area from forest to grassland and shrubland increased runoff volume by ∼200,000 m³ across increasing trend basins. Basins in the southern region of CONUS were more impacted by runoff parameters (RC and runoff volume) from directional land cover changes than basins in the northern region. The findings of this study are useful for planning and managing water availability for sustainable and adaptive water resources management at regional scales.