Quantifying the spatial variability of species-specific tree transpiration across hillslopes is important for estimating watershed-scale evapotranspiration (ET) and predicting spatial drought effects on vegetation. The objectives of this study are to (1) assess sap flux density (J(s)) and tree-level transpiration (T-s) across three contrasting zones a (riparian buffer, mid-hillslope and upland-hillslope, (2) determine how species-specific J(s) responds to vapour pressure deficit (VPD) and (3) estimate watershed-level transpiration (T-w) using T-s derived from each zone. During 2015 and 2016, we measured J(s) in eight tree species in the three topographic zones in a small 12-ha forested watershed in the Piedmont region of central North Carolina. In the dry year of 2015, loblolly pine (Pinus taeda), Virginia pine (Pinus virginiana) and sweetgum (Liquidambar styraciflua) J(s) rates were significantly higher in the riparian buffer when compared to the other two zones. In contrast, J(s) rates in tulip poplar (Liriodendron tulipifera) and red maple (Acer rubrum) were significantly lower in the buffer than in the mid-hillslope. Daily T-s varied by zone and ranged from 10 to 93 L/day in the dry year and from 9 to 122 L/day in the wet year (2016). J(s) responded nonlinearly to VPD in all species and zones. Annual T-w was 447, 377 and 340 mm based on scaled-J(s) data for the buffer, mid-hillslope and upland-hillslope, respectively. We conclude that large spatial variability in J(s) and scaled T-w was driven by differences in soil moisture at each zone and forest composition. Consequently, spatial heterogeneity of vegetation and soil moisture must be considered when accurately quantifying watershed level ET.