The transmission of information from DNA to RNA is a critical process. We compared RNA sequences from human B cells of 27 individuals to the corresponding DNA sequences from the same individuals and uncovered more than 10,000 exonic sites where the RNA sequences do not match that of the DNA. All 12 possible categories of discordances were observed. These differences were nonrandom as many sites were found in multiple individuals and in different cell types, including primary skin cells and brain tissues. Using mass spectrometry, we detected peptides that are translated from the discordant RNA sequences and thus do not correspond exactly to the DNA sequences. These widespread RNA-DNA differences in the human transcriptome provide a yet unexplored aspect of genome variation.