Lipoxygenase (LOX) is a ubiquitous oxygenase found in animals and plants and plays a pivotal role in diverse biological processes, including defense and development. Artemisinin, which can only be obtained from Artemisia annua L., is the most effective therapeutic drug for malaria without serious side effects. This study identified and analyzed LOX gene family members in the A. annua genome at the chromosomal level. Twenty LOX genes with various molecular weights, isoelectric points, and amino acid numbers were identified and named AaLOX, which were located in the cytoplasm or chloroplast. The average protein length of all AaLOX was 850 aa. Phylogenetic tree analysis revealed that the AaLOX was divided into two major groups, 9-LOX and 13-LOX. The exon numbers ranged from 1 to 12, indicating that different AaLOX genes have different functions. The secondary structure was mainly composed of alpha helix and random coil, and the tertiary structure was similar for most AaLOX. Upstream promoter region analysis revealed that a large number of cis-acting elements were closely related to plant growth and development, light response, hormone, and other stress responses. Transcriptome data analysis of different tissues suggested that the gene family was differently expressed in the roots, stems, leaves, and flowers of two A. annua strains HAN1 and LQ9. qRT-PCR confirmed that AaLOX5 and AaLOX17 had the highest expression in flowers and leaves. This study provides a theoretical basis for the further functional analysis of the AaLOX gene family.