tRFs are 14-32 base long single-stranded RNA derived from mature or precursor tRNA and are distinct from the stress-induced tRNA fragments created by cleavage in the anti-codon loop. tRFs have been shown to be important for cell-cycle progression and for regulating the dynamics of RISC. We report a comprehensive database of tRFs prepared from publicly available high-throughput sequencing data of >50 short RNA libraries. tRFs originate precisely from the extreme 5' (tRF-5) or 3' ends (tRF-3) of mature tRNAs or from the 3' trailer sequence of precursor tRNA transcripts (tRF-1) and are present in humans, mice, flies, worms and yeasts. tRF-3s are generated by cleavage of mature tRNA between A/U-A/U bases in the T?C loop. Over 150 tRFs are present in human and mouse cells at >20 reads/million, a frequency significantly higher than most microRNAs. Dicer and DGCR8 are dispensable for tRF generation, and >98% of the total tRF are not associated with Argonaute proteins Ago-1 or -2. As expected, tRF-5s and -3s are conserved in sequence across species because they are derived from functionally conserved mature tRNAs. Surprisingly, several tRF-1s are conserved in sequence between mice and humans, expressed in a tissue-specific manner and are highly abundant in embryos and embryonic stem cells. Specific tRF-1s are 100x induced in B cell malignancies compared to normal B cells, suggesting that they could serve as a biomarker for these malignancies. This database and the uniform nomenclature of tRFs will facilitate future investigations of this novel class of short RNAs.