Non-target-site-based resistance (NTSR) includes all types of mechanisms conferring resistance to herbicides that do not involve modifications at the herbicide target site. NTSR can be the most widespread type of resistance in a weed species, especially in grasses. NTSR can confer resistance to herbicides with different modes of action. Despite the threat it sets upon chemical weed control, NTSR is still the dark side of resistance to herbicides. In particular, NTSR genetics is poorly known. As a consequence, there are no quick, molecular-based, NTSR diagnostic tools, which hampers the development of tactics aiming at delaying NTSR selection in the field. The lack of data regarding NTSR genetics essentially comes from the complex nature of NTSR. NTSR is basically a subset of the mechanisms governing weed response to the abiotic stress exerted by herbicide applications. As such, NTSR is a quantitative trait that is endowed by the regulation of the expression of a range of genes, and/or by the presence of specific alleles in the resistant weed plant genomes. The role played by several families of enzymes (e.g. cytochromes P450, glutathione-S-transferases, esterases, glycosyl-transferases, transporters) in the protection of resistant weed plants against herbicide action is well established, but the vast majority of the corresponding genes is currently unknown. Furthermore, none of the genes involved in the regulation of NTSR is known to date. Thus, the major challenge for weed scientists dealing with herbicide resistance is to elucidate the genetic bases of NTSR. The recent rise of ‘next generation’ sequencing and proteomics technologies now allows us to relatively easily and cheaply identify sets of genes associated with a phenotype among the thousands expressed in one plant. NTSR genes can be identified this way, provided an adequate procedure is subsequently implemented to unambiguously demonstrate their role in NTSR.