SummaryHerbicide resistance in grass weeds is now one of the greatest threats to sustainable cereal production in Northern Europe. Multiple‐herbicide resistance (MHR), a poorly understood multigenic and quantitative trait, is particularly problematic as it provides tolerance to most classes of chemistries currently used for post‐emergence weed control. Using a combination of transcriptomics and proteomics, the evolution of MHR in populations of the weed blackgrass (Alopecurus myosuroides) has been investigated. While over 4500 genes showed perturbation in their expression in MHR versus herbicide sensitive (HS) plants, only a small group of proteins showed >2‐fold changes in abundance, with a mere eight proteins consistently associated with this class of resistance. Of the eight, orthologues of three of these proteins are also known to be associated with multiple drug resistance (MDR) in humans, suggesting a cross‐phyla conservation in evolved tolerance to chemical agents. Proteomics revealed that MHR could be classified into three sub‐types based on the association with resistance to herbicides with differing modes of action (MoA), being either global, specific to diverse chemistries acting on one MoA, or herbicide specific. Furthermore, the proteome of MHR plants were distinct from that of HS plants exposed to a range of biotic (insect feeding, plant–microbe interaction) and abiotic (N‐limitation, osmotic, heat, herbicide safening) challenges commonly encountered in the field. It was concluded that MHR in blackgrass is a uniquely evolving trait(s), associated with changes in the proteome that are distinct from responses to conventional plant stresses, but sharing common features with MDR in humans.