R-loops are stable chromatin structures comprising a DNA:RNA hybrid and a displaced single-stranded DNA. R-loops have been implicated in gene expression and chromatin structure, as well as in replication blocks and genome instability. Here, we conducted a genome-wide identification of R-loops and identified more than 700,000 R-loop peaks in the maize (Zea mays) genome. We found that sense R-loops were mainly enriched in promoters and transcription termination sites, and relatively less enriched in gene bodies, which is different from the main gene-body localization of sense R-loops in Arabidopsis and Oryza sativa. At the chromosome scale, maize R-loops were enriched in pericentromeric heterochromatin regions and a significant portion of R-loops were derived from transposable elements. In centromeres, R-loops preferentially formed within the binding regions of the centromere-specific histone CENH3 and centromeric retrotransposons were strongly associated with R-loop formation. Furthermore, centromeric retrotransposon R-loops were observed by applying the single-molecule imaging technique of atomic force microscopy. These findings elucidate the fundamental character of R-loops in the maize genome and reveal the potential role of R-loops in centromeres.