Background <p>Organelle genome stability is essential for plant development and adaptation. Recombination of repetitive sequences in plant mitochondrial genomes is a key factor influencing this stability and is tightly regulated by nuclear-encoded proteins, including plant-specific organellar single-stranded DNA-binding (OSB) proteins. However, the evolutionary history and overall impact of <i>OSB</i> genes on organellar genome stability remain poorly understood.</p> Results <p>Here, we identified 182 <i>OSB</i> genes from 59 species across major plant clades. Seed plant <i>OSB</i> genes formed three phylogenetic groups with varying numbers of conserved PDF domains, while chlorophytes, streptophytes, bryophytes, lycophytes, ferns lay outside these groups with lower copies. Using high-fidelity (HiFi) long-read sequencing, we examined the effects of OSB deficiency on organellar genome stability in both eudicots (<i>Arabidopsis</i>) and monocots (rice). In <i>Arabidopsis osb1</i> and <i>osb4</i>, and rice <i>osb1</i> mutants, recombination increased at a subset of nearly identical mitochondrial medium-sized repeats but decreased at repeats longer than 1&#xa0;kb. These repeats displayed asymmetric accumulation of conformations, and a shift in the dominant genome conformation was also observed in some individuals. Moreover, different regions of the mutant mitochondrial genomes showed uneven HiFi read coverage.</p> Conclusions <p>Our findings highlight important roles of <i>OSB</i> genes in regulating the stability of plant mitochondrial genomes.</p>