Mapping land cover in highly heterogeneous landscapes is challenging, and classifications have inherent limitations where the spatial resolution of remotely sensed data exceeds the size of small objects. For example, classifications based on 30-m Landsat data do not capture urban or other heterogeneous environments well. This limitation may be overcome by quantifying the subpixel fractions of different land cover types. However, the selection process and transferability of models designed for subpixel land cover mapping across biomes is yet challenging. We asked to what extent (a) locally trained models can be used for sub-pixel land cover fraction estimates in other biomes, and (b) training data from different regions can be combined into spatially generalized models to quantify fractions across global biomes. We applied machine learning regression-based fraction mapping to quantify land cover fractions of 18 regions in five biomes using Landsat data from 2022. We used spectral-temporal metrics to incorporate intra-annual temporal information and compared the performance of local, spatially transferred, and spatially generalized models. Local models performed best when applied to their respective sites (average mean absolute error, MAE, 9–18%), and also well when transferred to other sites within the same biome, but not consistently so for out-of-biome sites. However, spatially generalized models that combined input data from many sites worked very well when analyzing sites in many different biomes, and their MAE values were only slightly higher than those of the respective local models. A weighted training data selection approach, preferring training data with a lower spectral distance to the image data to be predicted, further enhanced the performance of generalized models. Our results suggest that spatially generalized regression-based fraction models can support multi-class sub-pixel fraction estimates based on medium-resolution satellite images globally. Such products would have great value for environmental monitoring in heterogeneous environments and where land cover varies along spatial or temporal gradients.