Resistance to dicamba (a synthetic auxin herbicide) has been documented in Kochia scoparia (L.) Schrad. populations since 1994, but the molecular mechanisms of observed resistance cases remain elusive. An RNA-Seq approach was used to identify transcripts with significantly differential transcription responses between inbred lines of dicamba-resistant (9425R) and dicamba-susceptible (7710S) K. scoparia in response to dicamba application. Among the significantly differentially expressed transcripts was both Chalcone Synthase (CHS), the first enzyme and rate-limiting step in the flavonoid biosynthesis pathway, and Flavono 3'-Hydroxylase (F3'H), which catalyzes the conversion of quercetin into kaempferol, known inhibitors of auxin transport. In silico expression patterns of both transcripts were confirmed with qRT-PCR. An F2 population derived from a cross of 9425R x 7710S segregating for the resistance phenotype was assayed for CHS and F3'H expression using qRT-PCR. Dicamba-resistant F2 individuals displayed significantly higher CHS transcript abundance compared to dicamba-susceptible F2 individuals, associating the resistance phenotype of 9425R with a greater overall flux through the flavonoid biosynthesis pathway. Increased production of the auxin transport inhibitors quercetin and kaempferol could reduce intercellular transport and vascular loading of dicamba, causing a substantial reduction in dicamba efficacy by reducing its translocation to sensitive meristematic tissue, thereby conferring the observed resistance phenotype.