Background: Artemisia is a large and complex genus comprising about 500 species. Currently, only a limited number of plastomes (the chloroplast genome) of Artemisia are available. Their structures have not been comparatively analyzed, and a phylogenetic backbone based on plastome-scale data is still lacking. This situation has greatly hindered our understanding of the plastome variation patterns and infra-generic relationships of the genus. Methods: We newly sequenced 34 Artemisia plastomes representing 30 species and three varieties. Combining this with previously published plastomes, we comparatively analyzed their structure and constructed phylogenetic relationships using the protein-coding sequences (CDS) of plastomes. Results: Our analyses indicated that the Artemisia plastomes are conserved in terms of their structure, GC content, gene number, and order. The sequence divergence is higher in the LSC and SSC regions than in the IR regions. Three protein-coding genes and four non-coding regions, i.e., accD, petG, ycf1, rpoC1-rpoC2, rpoC2-rps2, trnG(UCC)-trnfM(CAU), and ndhG-ndhI, were highly diverse and could be chosen as candidates of DNA barcodes. Phylogenetic trees were divided into several clades, and all four main subgenera were not monophyletic. Additionally, the phylogenetic position of A. stracheyi is still controversial. Conclusions: Plastomes can provide important information for phylogenetic constructions. This study provides insights into the infra-generic relationships within Artemisia and also lays a foundation for future evolutionary studies of this genus.