Artemisia annua is the major natural source of artemisinin, an anti-malarial medicine commonly used worldwide. Here, we present chromosome-level haploid maps for two A. annua strains with different arte-misinin contents to explore the relationships between genomic organization and artemisinin production. High-fidelity sequencing, optical mapping, and chromatin conformation capture sequencing were used to assemble the heterogeneous and repetitive genome and resolve the haplotypes of A. annua. Approxi-mately 50,000 genes were annotated for each haplotype genome, and a triplication event that occurred approximately 58.12 million years ago was examined for the first time in this species. A total of 3,903,467???5,193,414 variants (SNPs, indels, and structural variants) were identified in the 1.5-Gb genome during pairwise comparison between haplotypes, consistent with the high heterozygosity of this species. Genomic analyses revealed a correlation between artemisinin concents and the copy number of amorpha-4,11-diene synthase genes. This correlation was further confirmed by resequencing of 36 A. annua samples with varied artemisinin contents. Circular consensus sequencing of transcripts facilitated the detection of paralog expression. Collectively, our study provides chromosome-level allele-aware genome assemblies for two A. annua strains and new insights into the biosynthesis of artemisinin and its regulation, which will contribute to conquering malaria worldwide.