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Abstract Papaver setigerum is an annual herb that is closely related to the opium poppy, P. somniferum. Genetic resources for P. setigerum are scarce. In the present study, we assembled the complete chloroplast (cp) genome of P. setigerum based on genome skimming data, and we conducted comparative cp genome analyses to study the evolutionary pattern in Papaveraceae. The cp genome of P. setigerum is 152,862 bp in length with a typical quadripartite structure. Comparative analyses revealed no gene rearrangement in the Papaveraceae family, although differences were evident in genome size, gene losses, as well as inverted repeats (IR) region expansion and contraction. The rps15 gene has been lost from the genomes of Meconopsis racemosa, Coreanomecon hylomeconoides, P. orientale, P. somniferum, and P. setigerum, and the ycf15 gene is found only in C. hylomeconoides. Moreover, 13 cpDNA markers, including psbA-trnH, rps16-trnQ, trnS-trnG, trnC-petN, trnE-trnT, trnL-trnF, trnF-ndhJ, petA-psbJ, ndhF-rpl32, rpl32-trnL, ccsA-ndhD, ndhE-ndhG, and rps15-ycf1, were identified with relatively high levels of variation within Papaver, which will be useful for species identification in this genus. Among those markers, psbA-trnH is the best one to distinguish P. somniferum and P. setigerum.