Metabolite composition and concentrations in seed grains are important traits of cereals. To identify the variation in the seed metabolotypes of a model grass, namely <i>Brachypodium distachyon</i>, we applied a widely targeted metabolome analysis to forty inbred lines of <i>B. distachyon</i> and examined the accumulation patterns of 183 compounds in the seeds. By comparing the metabolotypes with the population structure of these lines, we found signature metabolites that represent different accumulation patterns for each of the three <i>B. distachyon</i> subpopulations. Moreover, we found that thirty-seven metabolites exhibited significant differences in their accumulation between the lines Bd21 and Bd3-1. Using a recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21, we identified the quantitative trait loci (QTLs) linked with this variation in the accumulation of thirteen metabolites. Our metabolite QTL analysis illustrated that different genetic factors may presumably regulate the accumulation of 4-pyridoxate and pyridoxamine in vitamin B6 metabolism. Moreover, we found two QTLs on chromosomes 1 and 4 that affect the accumulation of an anthocyanin, chrysanthemin. These QTLs genetically interacted to regulate the accumulation of this compound. This study demonstrates the potential for metabolite QTL mapping in <i>B. distachyon</i> and provides new insights into the genetic dissection of metabolomic traits in temperate grasses.