Abstract Background and Aims It is accepted that contemporary allopolyploid species have originated recurrently, but very few cases have been documented using multiple natural formations of the same species. To extend our knowledge, we have investigated the multiple origins, genetic variation, and structure of the allotetraploid grass Brachypodium hybridum with respect to its progenitor diploid species B. distachyon (D genome) and B. stacei (S genome). For this, our primary focus is the Iberian Peninsula, an evolutionary hotspot for the genus Brachypodium. Methods We analysed 342 B. hybridum individuals from 36 populations using 10 nuclear SSR loci and two plastid loci. The B. hybridum genetic profiles were compared with those previously reported for B. stacei and B. distachyon. In addition, phylogenetic analysis of the plastid data was performed for a reduced subset of individuals. Key Results The nuclear SSR genetic analysis detected medium to high genetic diversity, with a strong south-to-north genetic structure cline, and a high selfing rate in B. hybridum. Comparative genetic analysis showed a close relatedness of current B. hybridum D allelic profiles with those of B. distachyon, but a lack of similarity with those of B. stacei, suggesting another B. stacei source for the B. hybridum S alleles. Plastid analysis detected three different bidirectional allopolyploidization events: two involved distinct B. distachyon-like ancestors and one involved a B. stacei-like ancestor. The Southeastern (SE) Iberian Peninsula B. hybridum populations were more genetically diverse and could have originated from at least two hybridization events whereas Northeaster-Northwestern (NE-NW) Iberian Peninsula B. hybridum populations were less diverse and may have derived from at least one hybridization event. Conclusions The genetic and evolutionary evidence support the plausible in situ origin of the SE and northern Iberian Peninsula B. hybridum allopolyploids from their respective local B. distachyon and unknown B. stacei ancestors. The untapped multiple origins and genetic variation detected in these B. hybridum populations opens the way to future evolutionary analysis of allopolyploid formation and genomic dominance and expression in the B. hybridum – B. distachyon – B. stacei grass model complex.