ABSTRACT The complement of tRNA genes within a genome is typically considered to be a stable characteristic of an organism. Here we demonstrate that bacterial tRNA gene set composition can be more flexible than previously appreciated, particularly regarding tRNA gene copy number. We report the spontaneous, high-rate occurrence of large-scale, tandem duplication events in laboratory populations of the bacterium Pseudomonas fluorescens SBW25. The identified duplication fragments are up to ~1 Mb in size (~15 % of the wildtype genome) and are predicted to change the copy number of up to 917 genes, including several tRNA genes. The observed duplication fragments are inherently unstable: they occur, and are subsequently lost, at extremely high rates. We propose that this unusually plastic type of mutation provides a mechanism by which tRNA gene set diversity can be rapidly generated, while simultaneously preserving the underlying tRNA gene set in the absence of continued selection. That is, if a tRNA set variant provides no fitness advantage, then high-rate segregation of the duplication fragment ensures the maintenance of the original tRNA gene set. However, if a tRNA gene set variant is beneficial, the underlying duplication fragments persist and provide the raw material for further, more stable, evolutionary change. REPOSITORY CONTENTS This entry contains additional raw data and background information for the above manuscript, organized into the following folders: background: this folder contains the background files for several experiments (colony images, gel images, Sanger sequences). data: this folder contains .xlsx sheets with the raw data, calculations, and statistics for several experiments (competition assays, genotype and population growth curves, duplication junction PCR assays, stability assays, YAMAT-seq sample preparation).