Although conventional immunohistochemistry for neurotropic Rabies virus (RABV) usually shows a high preference for neurons, non-neuronal cells are also potential target cells and abortive infection of astrocytes is considered a main trigger of innate immunity in the CNS. While in vitro studies indicated differences between field and less virulent lab-adapted RABVs, a systematic and quantitative comparison of astrocyte tropism in vivo is lacking. Here, a recently developed solvent-based tissue clearing technique was used to measure the RABV cell tropism in infected brains. Immunofluorescence analysis of 1 mm-thick tissue slices enabled 3D segmentation and quantification of infection frequencies of astrocytes and neurons. Comparison of highly virulent street virus clones from fox, dog, and raccoon with three lab strains of intermediate and low virulence revealed remarkable differences in the ability to infect astrocytes in vivo. While all viruses and infection routes led to comparable neuron infection frequencies, striking differences were detected for the infection of astrocytes. Consistent and inoculation route-independent astrocyte infection by field viruses, together with route-dependent or undetectable astrocyte infection by lab-adapted or vaccine viruses strongly suggests a model in which the ability to establish productive astrocyte infection in vivo functionally distinguishes field and attenuated lab RABV strains.