Accurate transmission and expression of genetic information are crucial for the survival of all living organisms. Recently, the coupling of mutation accumulation experiments and next-generation sequencing has greatly expanded our knowledge of the genomic mutation rate in both prokaryotes and eukaryotes. However, because of their transient nature, transcription errors have proven extremely difficult to quantify, and current estimates of transcription fidelity are derived from artificial constructs applied to just a few organisms. Here we report a unique cDNA library preparation technique that allows error detection in natural transcripts at the transcriptome-wide level. Application of this method to the model organism Caenorhabditis elegans revealed a base misincorporation rate in mRNAs of ~4 × 10(-6) per site, with a very biased molecular spectrum. Because the proposed method is readily applicable to other organisms, this innovation provides unique opportunities for studying the incidence of transcription errors across the tree of life.