Transposable elements (TEs) account for up to 80% of the wheat genome and are considered one of the main drivers of wheat genome evolution. However, the contribution of TEs to the divergence and evolution of wheat genomes is not fully understood. In this study, we have developed 55 miniature inverted-repeat transposable element (MITE) markers that are based on the presence/absence of an element, with over 60% of these 55 MITE insertions associated with wheat genes. We then applied these markers to assess genetic diversity among Triticum and Aegilops species, including diploid (AA, BB and DD genomes), tetraploid (BBAA genome) and hexaploid (BBAADD genome) species. While 18.2% of the MITE markers showed similar insertions in all species indicating that those are fossil insertions, 81.8% of the markers showed polymorphic insertions among species, subspecies, and accessions. Furthermore, a phylogenetic analysis based on MITE markers revealed that species were clustered based on genus, genome composition, and ploidy level, while 47.13% genetic divergence was observed between the two main clusters, diploids versus polyploids. In addition, we provide evidence for MITE dynamics in wild emmer populations. The use of MITEs as evolutionary markers might shed more light on the origin of the B-genome of polyploid wheat.