The water use by short rotation coppices (SRC) has been a focus of ongoing research in the last decades. Nevertheless, investigations that consider site factors and present long-term monitoring of the components of the water balance are rare. This research quantified the tree-based transpiration in the 4th growing season of uncoppiced 1st rotational hybrid poplar stands (Populus deltoides Bart. ex Marsh × P. nigra L. “AF2”) in western Slovakia. The aim of the study was to determine the influence of meteorological and soil-related site conditions on transpiration rates. Three experimental plots were located in the Morava River floodplains, on loamy sand-textured soils with different groundwater accessibilities: higher, low, and fluctuating groundwater level. We measured sap flow (Heat Ratio Method), volumetric water content, matric potential, groundwater level, and meteorological variables throughout the growing season in 2019. The results indicated that transpiration in the three sites was almost constant during that period, which was characterized by distinct conditions. The average cumulative transpiration at the site with a higher groundwater level (1105 mm) was larger than at the site with a lower groundwater level (632 mm) and the site with fluctuating groundwater (863 mm). A principal component analysis (PCA) and correlation analysis identified that the contribution of meteorological and soil-related site variables to transpiration differed among the sites. Soil water availability and groundwater accessibility are critical variables for the water use of poplar SRC. We concluded that the combination of site conditions needs to be reconsidered for the expansion of sustainable short rotation plantations in Europe.