This study investigated the roles of jasmonates in the regulation of sorgoleone accumulation and the expression of genes involved in sorgoleone biosynthesis in sorghum roots. Both methyl jasmonate (MeJa) and jasmonic acid (JA) substantially promoted root hair formation, secondary root development, root weight, and sorgoleone accumulation in sorghum roots. Sorgoleone content varied widely depending on the concentration of JA or MeJa and the duration of their application. Root weight and sorgoleone accumulation were highest after the application of JA or MeJa at a concentration of 5.0 μM, and then declined with increasing concentrations of jasmonates. At 5.0 μM, JA and MeJa increased sorgoleone content by 4.1 and 3.4-fold, respectively. Transcript accumulation was apparent for all genes, particularly for the O-methyltransferase 3 gene, which increased in expression levels up to 8.1-fold after a 36-h exposure to MeJa and 3.5-fold after a 48-h exposure to JA. The results of this study pave the way for more effective biosynthesis of sorgoleone, an important and useful allelochemical obtained from a variety of plant species.