Non-B-form DNA differs from the classic B-DNA double helix structure and plays a crucial regulatory role in replication and transcription However, the role of non-B-form DNA in centromeres, especially in polyploid wheat remains elusive. Here, we systematically analyzed seven non-B-form DNA motif profiles in hexaploid wheat, comprising A-phased DNA repeat, Direct repeat, G-quadruplex, Inverted Repeat, Mirror Repeat, Short Tandem Repeat and Z-DNA, in hexaploid wheat. We found that three of these non-B-form DNA motifs were enriched at centromeric regions, especially at the CENH3-binding sites, suggesting non-B-form DNA may create a favorable loading environment for the CENH3 nucleosome. To investigate the dynamics of centromeric non-B form DNA during alloploidization process, we analyzed DNA secondary structure using CENH3 ChIP-seq data form the newly formed allotetraploid wheat and its two diploid ancestors. We found newly formed allotetraploid wheat tended to form more non-B-form DNA in centromeric regions compared to their parents, suggesting non-B-form DNA is related to the localization of the centromeric regions in newly formed wheat. Furthermore, non-B-form DNA enriched in the centromeric regions were found to preferentially form on young LTR retrotransposons, explaining CENH3’s tendency to bind younger LTR. Collectively, our study provides the landscape of non-B-form DNA in the wheat genome, and sheds light on its potential role in the evolution of polyploid centromeres.