Summary Globally, weedy plants are a major constraint to sustainable crop production. Much of the success of weeds rests with their ability to rapidly adapt in the face of human‐mediated management of agroecosystems. Alopecurus myosuroides (blackgrass) is a widespread and impactful weed affecting agriculture in Europe. Here we report a chromosome‐scale genome assembly of blackgrass and use this reference genome to explore the genomic/genetic basis of non‐target site herbicide resistance (NTSR). Based on our analysis of F2 seed families derived from two distinct blackgrass populations with the same NTSR phenotype, we demonstrate that the trait is polygenic and evolves from standing genetic variation. We present evidence that selection for NTSR has signatures of both parallel and non‐parallel evolution. There are parallel and non‐parallel changes at the transcriptional level of several stress‐ and defence‐responsive gene families. At the genomic level, however, the genetic loci underpinning NTSR are different (non‐parallel) between seed families. We speculate that variation in the number, regulation and function of stress‐ and defence‐related gene families enable weedy species to rapidly evolve NTSR via exaptation of genes within large multi‐functional gene families. These results provide novel insights into the potential for, and nature of plant adaptation in rapidly changing environments.