Terrestrial plant biomass is a key biophysical parameter required for understanding ecological systems in Alaska. An accurate estimation of biomass at a regional scale provides an important data input for ecological modeling in this region. In this study, we created an aboveground biomass (AGB) map at 30-m resolution for the Yukon Flats ecoregion of interior Alaska using Landsat data and field measurements. Tree, shrub, and herbaceous AGB data in both live and dead forms were collected in summers and autumns of 2009 and 2010. Using the Landsat-derived spectral variables and the field AGB data, we generated a regression model and applied this model to map AGB for the ecoregion. A 3-fold cross-validation indicated that the AGB estimates had a mean absolute error of 21.8 Mg/ha and a mean bias error of 5.2 Mg/ha. Additionally, we validated the mapping results using an airborne lidar dataset acquired for a portion of the ecoregion. We found a significant relationship between the lidar-derived canopy height and the Landsat-derived AGB (R2 = 0.40). The AGB map showed that 90% of the ecoregion had AGB values ranging from 10 Mg/ha to 134 Mg/ha. Vegetation types and fires were the primary factors controlling the spatial AGB patterns in this ecoregion.