Weeds in the Lolium genus, notably, Lolium rigidum and Lolium multiflorum, significantly threaten global crop yields and food production due to evolved glyphosate resistance from heavy reliance on this herbicide. However, the molecular basis for non-target site resistance (NTSR) remains unclear. Comprehensive genomic and transcriptomic analyses were performed on glyphosate-resistant and -susceptible biotypes of L. rigidum and L. multiflorum to identify key genes and regulatory networks linked to this resistance. Several key gene families were associated with herbicide detoxification: ABC transporters (ABCs), cytochrome P450s (CYP450s), aldo-keto reductases (AKRs), and glutathione S-transferases (GSTs). Notably, ABCC3 emerged as a potential conserved NTSR gene in Lolium spp. Transgenic rice overexpressing LrABCC3 exhibited enhanced glyphosate resistance, while candidate transcription factors potentially regulating ABCC3 were identified, with LrB3 being validated to activate its promoter. This study identified ABCC3 as the first conserved NTSR gene linked to glyphosate resistance in Lolium weeds.