R-loops are three-stranded structures consisting of a DNA:RNA hybrid and a single-stranded DNA, which play essential biological roles in eukaryotes. However, their functions during hybridization and the long-term evolutionary trajectory of polyploidization remain unclear. Here, we compared R-loops profiles between the allotetraploid Brachypodium hybridum and its two diploid progenitors, B. distachyon and B. stacei. Our results revealed that R-loops are highly conserved in syntenic regions between the diploid and allotetraploid genomes. Moreover, their formation is strongly sequence-dependent and is stably maintained even in regions that have undergone structural rearrangement such as translocations and inversions following polyploidization. These sequence-dependent R-loops were largely unaffected by RNA abundance or chromatin accessibility but were associated with small sequence variations such as SNPs, deletions, and insertions. Notably, we observed global increases and decreases in CG, CHG, and CHH DNA methylation levels at sites of upregulated and downregulated R-loops, respectively. Both sense and antisense DNA strands were affected by fluctuations of R-loop abundance. Collectively, these findings reveal that R-loop formation and dynamics during polyploidization are sequence-directed and coupled with both genetic variation and epigenetic reprogramming, providing new insights into their roles in shaping plant genome structure.