Abstract G-quadruplexes are nucleic acid secondary structures formed by a stack of square planar G-quartets. G-quadruplexes were implicated in many biological functions including telomere maintenance, replication, transcription, and translation, in many species including humans and plants. For wheat, however, though it is one of the world’s most important staple food, no G-quadruplex studies have been reported to date. Here, we computationally identify putative G4 structures (G4s) in wheat genome for the first time and compare its distribution across the genome against five other genomes (human, maize, Arabidopsis, rice, and sorghum). We identified close to 1 million G4 motifs with a density of 76 G4s/Mb across the whole genome and 93 G4s/Mb over genic regions. Remarkably, G4s were enriched around three regions, two located on the antisense and one on the sense strand at the following positions: 1) the transcription start site (TSS) (antisense), 2) the first coding domain sequence (CDS) (antisense), and 3) the start codon (sense). Functional enrichment analysis revealed that the gene models containing G4 motifs within these peaks were associated with specific gene ontology (GO) terms, such as developmental process, localization, and cellular component organization or biogenesis. We investigated genes encoding MADS-box transcription factors and showed examples of G4 motifs within critical regulatory regions in the VRN-1 genes in wheat. Furthermore, comparison with other plants showed that monocots share a similar distribution of G4s, but Arabidopsis shows a unique G4 distribution. Our study shows for the first time the prevalence and possible functional roles of G4s in wheat.