Quantifying species-specific tree transpiration across watershed zones is important for estimating watershed evapotranspiration (ET) and predicting drought effects on vegetation. The objectives of this study are to 1) assess sap flux density (Js) and tree-level transpiration (Ts) across three contrasting zones (riparian buffer, mid-hillslope, and upland-hillslope), 2) determine how species-specific Js responds to vapor pressure deficit (VPD), and 3) compare watershed-level transpiration (Tw) derived from each zone. We measured Js and Ts in eight tree species in the three zones in a 12-ha forested watershed. In the dry year of 2015, loblolly pine (Pinus taeda), Virginia pine (Pinus virginiana), and sweetgum (Liquidambar styraciflua) Js rates were significantly higher in the buffer when compared to the other two zones. In contrast, Js in tulip poplar (Liriodendron tulipifera) and red maple (Acer rubrum) were significantly lower in the buffer than in the mid-hillslope. Daily Ts varied by zone and ranged from 10 to 93 liters in the dry year and 9 to 122 liters in the wet year. Js responded nonlinearly to VPD in all trees and zones. Annual Tw based on scaled-Js data was 447 mm, 377 mm, and 340 mm for the buffer, mid-hillslope, and upland-hillslope, respectively. We conclude that large spatial variability in Js and scaled Tw were 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.