The process of mRNA vaccine manufacturing relies on proper DNA digestion following an in-vitro transcription reaction to remove residual contaminating DNA from the plasmid backbone from the process. To assess the quality and quantity of potential DNA impurities in mRNA vaccines, we analyzed unopened, cold-chain compliant vaccine lots for residual DNA contamination using quantitative PCR (qPCR), RNase A/Qβubit fluorometry, and Oxford Nanopore sequencing from two Pfizer and three Moderna vials. We compared spike-protein amplicons and plasmid-vector amplicons to distinguish between DNA contaminant as double stranded DNA (dsDNA) versus RNA:DNA hybrids, qPCR assays revealed more than a 100-fold discrepancy in quantitation between dsDNA with RNA:DNA hybrids consistent with uneven DNase I digestion efficiency during mRNA vaccine manufacturing. Indeed, treatment of vaccines with DNase I-XT resulted in 100-1000X higher degradation of spike DNA, particularly in plasmid regions that form RNA:DNA hybrids. Together these results indicate that residual DNA testing which relies on a single qPCR for dsDNA fails to accurately quantify impurities, and that treating vaccine preparations with DNase I-XT during the manufacturing process may improve the quality by reducing contamination due to RNA:DNA hybrids. Keywords: mRNA vaccines, residual DNA, RNA:DNA hybrids, DNase resistance, quality control, qPCR