The small, annual grass <
i>
Brachypodium distachyon<
/i>
(L.) Beauv., a close relative of wheat (<
i>
Triticum aestivum<
/i>
L.) and barley (<
i>
Hordeum vulgare<
/i>
L.), is a powerful model system for cereals and bioenergy grasses. Genome-wide association studies (GWAS) of natural variation can elucidate the genetic basis of complex traits but have been so far limited in <
i>
B. distachyon<
/i>
by the lack of large numbers of well-characterized and sufficiently diverse accessions. Here, we report on genotyping-by-sequencing (GBS) of 84 <
i>
B. distachyon<
/i>
, seven <
i>
B. hybridum<
/i>
, and three B. stacei accessions with diverse geographic origins including Albania, Armenia, Georgia, Italy, Spain, and Turkey. Over 90,000 high-quality single-nucleotide polymorphisms (SNPs) distributed across the Bd21 reference genome were identified. Our results confirm the hybrid nature of the <
i>
B. hybridum<
/i>
genome, which appears as a mosaic of <
i>
B. distachyon<
/i>
-like and <
i>
B. stacei<
/i>
-like sequences. Analysis of more than 50,000 SNPs for the <
i>
B. distachyon<
/i>
accessions revealed three distinct, genetically defined populations. Surprisingly, these genomic profiles are associated with differences in flowering time rather than with broad geographic origin. High levels of differentiation in loci associated with floral development support the differences in flowering phenology between <
i>
B. distachyon<
/i>
populations. Genome-wide association studies combining genotypic and phenotypic data also suggest the presence of one or more photoperiodism, circadian clock, and vernalization genes in loci associated with flowering time variation within <
i>
B. distachyon<
/i>
populations. As a result, our characterization elucidates genes underlying population differences, expands the germplasm resources available for <
i>
Brachypodium<
/i>
, and illustrates the feasibility and limitations of GWAS in this model grass.