A major challenge in studies estimating stand water use in mixed-species forests is how to effectively scale data from individual trees to the stand. This is the case for forest ecosystems in the northeastern USA where differences in water use among species and across different size classes have not been extensively studied, despite their relevance for a wide range of ecosystem services. Our objectives were to assess the importance of different sources of variability on transpiration upscaling and explore the potential impacts of future shifts in species composition on the forest water budget. We measured sap velocity in five tree species (Fagus grandifolia Ehrh., Acer rubrum L., Acer saccharum Marsh., Betula alleghaniensis Britton, and Betula papyrifera Marsh.) in a mature stand and a young stand in New Hampshire, USA. Our results showed that the greatest potential source of error was radial variability and that tree size was more important than species in determining sap velocity. Total sapwood area was demonstrated to exert a strong controlling influence on transpiration, varying depending on tree size and species. We conclude that the effect of potential species shifts on transpiration will depend on the sap velocity, determined not only by radial variation and tree size, but also by the sapwood area distribution in the stand.