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Short RNAs derived from the cleavage of tRNA molecules are observed in most organisms. Their occurrence seems to be induced by stress conditions, but still little is known about their biogenesis and functions. We find that the recovery of tRNA fragments depends on the RNA isolation method. Using an optimized RNA extraction protocol and northern blot hybridization technique, we show that the tRNA‐derived fragments in yeast are widespread in 12 different growth conditions. We did not observe significant stress‐dependent changes in the amounts of tRNA fragments pool. Instead, we show the differential processing of almost all individual tRNAs. We also provide evidence that 3′‐part‐derived tRNA fragments are as abundant as the 5′‐ one in Saccharomyces cerevisiae. The resulting set of S. cerevisiae tRNA fragments provides a robust basis for further experimental studies on biological functions of tRFs.