Long non-coding RNAs (lncRNAs) are crucial in regulating secondary metabolite production in plants, but their role in artemisinin (ART) biosynthesis, a key anti-malarial compound from Artemisia annua, remains unclear. Here, by investigating high-artemisinin-producing (HAP) and lowartemisinin-producing (LAP) genotypes, we found that the final artemisinin content in A. annua is influenced by the quantity of the precursor compounds. We report on RNA deep sequencing in HAP and LAP genotypes. Based on the application of a stringent pipeline, 1419 novel lncRNAs were identified. Moreover, we identified 256 differentially expressed lncRNAs between HAP and LAP. We then established correlations between lncRNAs and artemisinin biosynthesis genes in order to identify a molecular framework for the differential expression of the pathway between the two genotypes. Three potential lncRNAs (MSTRG.33718.2, MSTRG.30396.1 and MSTRG.2697.4) linked to the key artemisinin biosynthetic genes (ADS: Amorpha-4,11-diene synthase, DXS: 1-deoxy-D-xylulose-5-phosphate synthase, and HMGS: 3-hydroxyl-3-methyglutaryl CoA synthase) were detected. Importantly, we observed that up-regulation of these lncRNAs positively modulates the target artemisinin biosynthetic genes, potentially leading to high artemisinin biosynthesis in HAP. In contrast, BAS (beta-amyrin synthase), which is involved in the artemisinin competing pathway, was strongly down-regulated in HAP compared to LAP, in line with the expression pattern of the linked lncRNA MSTRG.30396.1. By identifying and characterizing lncRNAs that are potentially linked to the regulation of key biosynthetic genes, this work provides new insights into the complex regulatory networks governing artemisinin production in A. annua. Such findings could pave the way for innovative approaches in metabolic engineering, potentially enhancing artemisinin yields and addressing challenges in sustainable production.