Aberrant repair of DNA double-strand breaks can recombine distant chromosomal breakpoints. Chromosomal rearrangements compromise genome function and are a hallmark of ageing. Rearrangements are challenging to detect in non-dividing cell populations, because they reflect individually rare, heterogeneous events. The genomic distribution of de novo rearrangements in non-dividing cells, and their dynamics during ageing, remain therefore poorly characterized. Studies of genomic instability during ageing have focussed on mitochondrial DNA, small genetic variants, or proliferating cells. To characterize genome rearrangements during cellular ageing in non-dividing cells, we interrogated a single diagnostic measure, DNA breakpoint junctions, using Schizosaccharomyces pombe as a model system. Aberrant DNA junctions that accumulated with age were associated with microhomology sequences and R-loops. Global hotspots for age-associated breakpoint formation were evident near telomeric genes and linked to remote breakpoints elsewhere in the genome, including the mitochondrial chromosome. Formation of breakpoint junctions at global hotspots was inhibited by the Sir2 histone deacetylase and might be triggered by an age-dependent de-repression of chromatin silencing. An unexpected mechanism of genomic instability may cause more local hotspots: age-associated reduction in an RNA-binding protein triggering R-loops at target loci. This result suggests that biological processes other than transcription or replication can drive genome rearrangements. Notably, we detected similar signatures of genome rearrangements that accumulated in old brain cells of humans. These findings provide insights into the unique patterns and possible mechanisms of genome rearrangements in non-dividing cells, which can be promoted by ageing-related changes in gene-regulatory proteins. Author summary: DNA breaks followed by chromosomal rearrangements that join non-neighboring DNA sequences may critically affect gene function, evolution, and ageing. Such chromosomal rearrangements are difficult to spot in sequence data even if they are widespread, because they are individually rare and reflect diverse events. Here we establish sensitive analyses of DNA sequences and identify prevalent rearrangements that specifically accumulate during ageing in yeast cells. These rearrangements feature short repeated DNA sequences near the breaks, preferentially occur in certain locations of the chromosomes (e.g., near their ends), and can link sequences originating from different chromosomes. We show results indicating that DNA-RNA interactions, triggered by the ageing-associated suppression of an RNA-binding protein, can cause the non-random patterns of some chromosomal rearrangements. Our analyses suggest that similar patterns of chromosomal rearrangements accumulate in brain cells in older humans, raising the possibility that such DNA changes occurring in ageing cells are conserved from yeast to human. [ABSTRACT FROM AUTHOR]