摘要An R-loop is a three-stranded chromatin struc-ture that consists of a displaced single strand of DNA and an RNA:DNA hybrid duplex, which was thought to be a rare by-product of tran-scription. However, recent genome-wide data have shown that R-loops are widespread and pervasive in a variety of genomes, and a growing body of experimental evidence in-dicates that R-loops have both beneficial and harmful effects on an organism. To maximize benefit and avoid harm, organisms have evolved several means by which they tightly regulate R-loop levels. Here, we summarize our current understanding of the biogenesis and effects of R-loops, the mechanisms that regu-late them, and methods of R-loop profiling, re-viewing recent research advances on R-loops in plants. Furthermore, we provide perspectives on future research directions for R-loop biology in plants, which might lead to a more compre-hensive understanding of R-loop functions in plant genome regulation and contribute to fu-ture agricultural improvements.