There are more than 100 different ribonucleoside structures incorporated as post-transcriptional modifications, mainly in tRNA and rRNA of both prokaryotes and eukaryotes, and emerging evidence suggests that these modifications function as a system in the translational control of cellular responses. However, our understanding of this system is hampered by the paucity of information about the complete set of RNA modifications present in individual organisms. To this end, we have employed a chromatography-coupled mass spectrometric approach to define the spectrum of modified ribonucleosides in microbial species, starting with Mycobacterium bovis BCG. This approach revealed a variety of ribonucleoside candidates in tRNA from BCG, of which 12 were definitively identified based on comparisons to synthetic standards and 5 were tentatively identified by exact mass comparisons to RNA modification databases. Among the ribonucleosides observed in BCG tRNA was one not previously described in tRNA, which we have now characterized as N6,N6-dimethyladenosine.