Infections with high-risk human papillomaviruses (HPV), mainly HPV type 16, can cause malignant transformation of the human cervical epithelium and the development of cervical cancer (CxCa). A rapid and precise diagnosis of the precancerous lesions by conventional cytology or HPV DNA tests remains difficult and often leads to overtreatment. We quantitatively analyzed the HPV16 transcriptome of 80 HPV16 DNA-positive cervical scrapes classified as mild cytologic grade, including no intraepithelial lesion or malignancy (NIL/M; normal, n=25) and low-grade squamous intraepithelial lesion (LSIL; n=24), and severe cytologic grade, including high-grade squamous intraepithelial lesion (HSIL; n=24) and CxCa (n=7), with novel nucleic acid sequence-based amplification-Luminex assays. In severe lesions, HPV16 E6*II and E1C encoding transcripts were strongly upregulated, whereas spliced E1[SYMBOL: SEE TEXT]E4 and L1 encoding transcripts were markedly downregulated. Using a combination of the four marker transcripts, 100% of CxCa and 67% of HSIL cases were correctly identified as severe, and 74% of LSIL and 92% of NIL/M samples as mild cytologic grade. Compared with a commercially available HPV E6/E7 mRNA assay, the specificity of the marker combination for discriminating severe and mild cytologic lesions increased from 23% to 83%. In conclusion, we identified a novel HPV16 RNA pattern for grading of cervical lesions with a potentially high diagnostic value for the primary screening of CxCa precursors and the triage of cervical lesions.