The rapid production of messenger RNA (mRNA)-based vaccines has been chosen as the most suitable strategy to fight the COVID-19 pandemic. Three studies reported the presence of DNA in significant amounts in Pfizer mRNA vaccines. We aimed to confirm the presence of this residual DNA. Vaccine plasmid DNA quantification using the Qubit fluorometer on a vaccine vial showed it was 216 ng/dose on average and approximately 24 times greater, reaching 5,160 ng/dose on average, after treatment with Triton-X-100. In addition, we obtained by next-generation sequencing the sequence of the complete plasmid DNA vaccine matrix (7,824 base pairs) with high coverage (98.3%) and sequencing depths (mean, 4,181-4,389 reads), indicating the presence of the plasmid DNA in high copy number. These results calls for an assessment of the copy number and nature of DNA in mRNA vaccines at a larger scale and in multiple batches, notably regarding the putative risk of DNA integration after delivery into cells.