Abstract Resistance to preemergence herbicides, e.g. inhibitors of the biosynthesis of very-long-chain fatty acids (VLCFAs), is evolving in response to increased use of these compounds. Grass weeds such as ryegrasses (Lolium spp.) have accumulated resistance to various herbicide modes of action. Here, an RNA-seq analysis was conducted using 3 ryegrass populations resistant to the VLCFA biosynthesis inhibitor flufenacet to investigate this phenomenon. Besides various transcripts, including putative long noncoding RNAs (lncRNAs), a single putatively functional tau class glutathione transferase (GST) was constitutively differentially expressed. It was further induced by herbicide application. This GST was expressed as a recombinant protein in Escherichia coli along with other GSTs and detoxified flufenacet rapidly in vitro. Detoxification rates of other herbicides tested in vitro were in accordance with cross-resistance patterns previously determined in vivo. A genome-wide GST analysis revealed that the candidate GST was located in a cluster of 3 intronless GSTs. Their intronless nature possibly results from the retroposition of cellular mRNAs followed by tandem duplication and may affect gene expression. The large number of GSTs (≥195) in the genome of rigid ryegrass (Lolium rigidum) compared with other plant organisms is likely a key factor in the ability of this weed to evolve resistance to different herbicide chemistries. However, in the case of flufenacet resistance, a single upregulated GST with high affinity for the substrate flufenacet possibly contributes overproportionally to rapid herbicide detoxification in planta. The regulation of this gene and the role of differentially expressed transcripts, including various putative lncRNAs, require further investigation.