Brazilian tropical ecosystems in the state of Mato Grosso have experienced significant land use and cover changes during the past few decades due to deforestation and wildfire. These changes can directly affect the mass and energy exchange near the surface and, consequently, evapotranspiration (ET). Characterization of the seasonal patterns of ET can help in understanding how these tropical ecosystems function with a changing climate. The goal of this study was to characterize temporal (seasonal-to-decadal) and spatial patterns in ET over Mato Grosso using remotely sensed products. Ecosystems over areas with limited to no flux towers can be performed using remote sensing products such as NASA’s MOD16A2 ET (MOD16 ET). As the accuracy of this product in tropical ecosystems is unknown, a secondary objective of this study was to evaluate the ability of the MOD16 ET (ETMODIS) to appropriately represent the spatial and seasonal ET patterns in Mato Grosso, Brazil. Actual ET was measured (ETMeasured) using eight flux towers, three in the Amazon, three in the Cerrado, and two in the Pantanal of Mato Grosso. In general, the ETMODIS of all sites had no significant difference from ETMeasured during all analyzed periods, and ETMODIS had a significant moderate to strong correlation with the ETMeasured. The spatial variation of ET had some similarity to the climatology of Mato Grosso, with higher ET in the mid to southern parts of Mato Grosso (Cerrado and Pantanal) during the wet period compared to the dry period. The ET in the Amazon had three seasonal patterns, a higher and lower ET in the wet season compared to the dry season, and minimal to insignificant variation in ET during the wet and dry seasons. The wet season ET in Amazon decreased from the first and second decades, but the ET during the wet and dry season increased in Cerrado and Pantanal in the same period. This study highlights the importance of deepening the study of ET in the state of Mato Grosso due to the land cover and climate change.