Quantifying spatially explicit or pixel-level aboveground forest biomass (AFB) across large regions is critical for measuring forest carbon sequestration capacity, assessing forest carbon balance, and revealing changes in the structure and function of forest ecosystems. When AFB is measured at the species level using widely available remote sensing data, regional changes in forest composition can readily be monitored. In this study, wall-to-wall maps of species-level AFB were generated for forests in Northeast China by integrating forest inventory data with Moderate Resolution Imaging Spectroradiometer (MODIS) images and environmental variables through applying the optimal k-nearest neighbor (kNN) imputation model. By comparing the prediction accuracy of 630 kNN models, we found that the models with random forest (RF) as the distance metric showed the highest accuracy. Compared to the use of single-month MODIS data for September, there was no appreciable improvement for the estimation accuracy of species-level AFB by using multi-month MODIS data. When k > 7, the accuracy improvement of the RF-based kNN models using the single MODIS predictors for September was essentially negligible. Therefore, the kNN model using the RF distance metric, single-month (September) MODIS predictors and k = 7 was the optimal model to impute the species-level AFB for entire Northeast China. Our imputation results showed that average AFB of all species over Northeast China was 101.98 Mg/ha around 2000. Among 17 widespread species, larch was most dominant, with the largest AFB (20.88 Mg/ha), followed by white birch (13.84 Mg/ha). Amur corktree and willow had low AFB (0.91 and 0.96 Mg/ha, respectively). Environmental variables (e.g., climate and topography) had strong relationships with species-level AFB. By integrating forest inventory data and remote sensing data with complete spatial coverage using the optimal kNN model, we successfully mapped the AFB distribution of the 17 tree species over Northeast China. We also evaluated the accuracy of AFB at different spatial scales. The AFB estimation accuracy significantly improved from stand level up to the ecotype level, indicating that the AFB maps generated from this study are more suitable to apply to forest ecosystem models (e.g., LINKAGES) which require species-level attributes at the ecotype scale.