A dicamba-resistant population of kochia (Bassia scoparia) previously identified in Colorado, USA in 2012 was used to generate a synthetic mapping population that segregated simply for dicamba resistance. Linkage mapping to associate injury following dicamba application with genotype information from restriction-site-associated DNA sequencing identified a single locus in the kochia genome associated with resistance on chromosome 4. A mutant version of Auxin/Indole-3-Acetic Acid 16 (AUX/IAA16; a gene previously implicated in dicamba resistance in kochia) is found near the middle of this locus in resistant plants. Long read sequencing of resistant plants identified a recently inserted Ty1/Copia retrotransposon near the beginning of exon two of AUX/IAA16, leading to interruption of normal splicing. Stable transgenic lines of Arabidopsis thaliana ectopically expressing the mutant and wildtype alleles of AUX/IAA16 were developed. Arabidopsis thaliana plants expressing the mutant AUX/IAA16 allele grew shorter roots on control media but had less reduction in root growth on media containing either dicamba (5 μM) or IAA (0.5 μM) compared to non-transgenic plants or those expressing the wildtype allele of AUX/IAA16. Protein modeling suggests the substitution of a specific glycine residue in the degron domain of AUX/IAA16 is especially important for resistance. A fitness cost associated with the mutant allele of AUX/IAA16 has implications for resistance evolution and management of kochia populations with this resistance mechanism. We report a molecular assay for rapid detection of the mutant AUX/IAA16 allele that can inform in-season management decisions of agricultural producers.