The oxygen isotope composition of evapotranspiration (δF) represents an important tracer in the study of biosphere–atmosphere interactions, hydrology, paleoclimate, and carbon cycling. Here, we demonstrate direct measurement of δF based on the eddy-covariance and tunable diode laser spectroscopy (EC-TDL) techniques. Results are presented from laboratory experiments and field measurements in agricultural ecosystems. The field measurements were obtained during the growing seasons of 2008 and 2009. Water vapour mixing ratios (χw) and fluxes (F) were compared using EC-TDL and traditional eddy-covariance and infrared gas analyser techniques over a soybean canopy in 2008. The results indicate that χw and F agreed to within 1 and 6%, respectively. Measurements of δF above a corn canopy in 2009 revealed a diurnal pattern with an expected progressive 18O enrichment through the day ranging from about −20‰ before sunrise to about −5‰ in late afternoon. The isotopic composition of evapotranspiration was similar to the xylem water isotope composition (δx = −7.2‰) for short periods of time during 1400–1800 LST, indicating near steady-state conditions. Finally, the isotopic forcing values (IF) revealed a diurnal pattern with mean maximum values of 0.09ms−1‰ at midday. The IF values could be described as an exponential relation of relative humidity confirming previous model calculations and measurements over a soybean canopy in 2006. These patterns and comparisons indicate that long-term continuous isotopic water vapour flux measurements based on the eddy-covariance technique are feasible and can provide new insights related to the oxygen isotope fractionation processes at the canopy scale.