Post-transcriptional regulation of gene expression contributes to the protein output of a cell, however, methods for measuring translational regulation in complex in vivo systems are lacking. Here, we describe a sensitive method for measuring translational regulation in defined cell populations from heterogeneous tissue in vivo. We adapted the translating ribosome affinity purification (TRAP) methodology to measure the relative occupancy of individual mRNA transcripts in translating ribosomes in the Olig2-positive tumor cell population in a genetically engineered mouse model (GEM) of glioma. Global measurement of paired ribosome-bound and total cellular mRNA populations from tumor cells in vivo identified a broad distribution of relative ribosome occupancies amongst mRNA species that was highly reproducible across biological samples. Comparison of the translation state of glioma cells to non-transformed oligodendrocyte progenitor cells in normal brain identified global alteration of translation in tumor, and specifically of genes involved in cell division and synthetic metabolism. Furthermore, investigation of alteration in steady state translational efficiencies upon loss of PTEN, one of the most frequently mutated and deleted tumor suppressors in glioma, identified differential translation of proteins involved in cellular respiration, canonically regulated by PI3K/Akt signaling, and cellular glycosylation profiles, deregulation of which is known to be associated with tumor progression. Application of the translation efficiency profiling method described here to other biological contexts and conditions would extend our knowledge of the scope and impact of this important mode of gene regulation in complex in vivo systems.