tRNA is the most heavily modified of all RNA types, with typically 10–20% of the residues being post–transcriptionally altered. Unravelling the modification pattern of a tRNA is a challenging task; there are 92 currently known tRNA modifications , many of which are chemically similar. Furthermore, the tRNA has to be investigated with single–nucleotide resolution in order to ensure complete mapping of all modifications. In the present work, we characterized tRNALys(UUU) from Trypanosoma brucei, and provide a complete overview of its post–transcriptional modifications. The first step was MALDI–TOF MS of two independent digests of the tRNA, with RNase A and RNase T1, respectively. This revealed digestion products harbouring mass–changing modifications. Next, the modifications were mapped at the nucleotide level in the RNase products by tandem MS. Comparison with the sequence of the unmodified tRNA revealed the modified residues. The modifications were further characterized at the nucleoside level by chromatographic retention time and fragmentation pattern upon higher–order tandem MS. Phylogenetic comparison with modifications in tRNALys from other organisms was used through the entire analysis. We identified modifications on 12 nucleosides in tRNALys(UUU), where U47 exhibited a novel modification, 3–(3–amino–3–carboxypropyl)–5,6–dihydrouridine, based on identical chromatographic retention and MS fragmentation as the synthetic nucleoside. A37 was observed in two versions: a minor fraction with the previously described 2–methylthio–N6–threonylcarbamoyl–modification, and a major fraction with A37 being modified by a 294.0–Da moiety. The latter product is the largest adenosine modification reported so far, and we discuss its nature and origin.