Understanding the seasonality of the transpiration fraction (T/ET) of total terrestrial evapotranspiration (ET) is vital for coupling ecological and hydrological systems and quantifying the heterogeneity among various ecosystems. In this study, a two‐source model was used to estimate T/ET in five ecosystems over the Heihe River Basin. In situ measurements of daily energy flux, sap flow, and surface soil temperature were compared with model outputs for 2014 and 2015. Agreement between model predictions and observations demonstrates good performance in capturing the ecosystem seasonality of T/ET. In addition, sensitivity analysis indicated that the model is insensitive to errors in measured input variables and parameters. T/ET among the five sites showed only slight interannual fluctuations while exhibited significant seasonality. All the ecosystems presented a single‐peak trend, reaching the maximum value in July and fluctuating day to day. During the growing season, average T/ET was the highest for the cropland ecosystem (0.80 ± 0.13), followed by the alpine meadow ecosystem (0.79 ± 0.12), the desert riparian forest Populus euphratica (0.67 ± 0.07), the Tamarix ramosissima Ledeb desert riparian shrub ecosystem (0.67 ± 0.06), and the alpine swamp meadow (0.55 ± 0.23). Leaf area index exerted a first‐order control on T/ET and showed divergence among the five ecosystems because of different vegetation dynamics and environmental conditions (e.g., water availability or vapor pressure deficits). This study quantified transpiration fraction across diverse ecosystems within the same water basin and emphasized the biotic controls on the seasonality of the transpiration fraction
