Experimentally characterizing evapotranspiration (ET) in different biomes around the world is an issue of interest for different areas of science. ET in natural areas of the Brazilian Pampa biome has still not been assessed. In this study, the actual ET (ETact) obtained from eddy covariance measurements over two sites of the Pampa biome was analyzed. The objective was to evaluate the energy partition and seasonal variability of the actual ET of the Pampa biome. Results showed that the latent heat flux was the dominant component in available energy in both the autumn–winter (AW) and spring–summer (SS) periods. Evapotranspiration of the Pampa biome showed strong seasonality, with highest ET rates in the SS period. During the study period, approximately 65% of the net radiation was used for the evapotranspiration process in the Pampa biome. The annual mean ET rate was 2.45 mm d−1. ET did not show to vary significantly between sites, with daily values very similar in both sites. The water availability in the Pampa biome was not a limiting factor for ET, which resulted in a small difference between the reference ET and the actual ET. These results are helpful in achieving a better understanding of the temporal pattern of ET in relation to the landscape of the Pampa biome and its meteorological, soil, and vegetation characteristics.