HPV genotyping is an important tool in the epidemiology and surveillance of HPV-associated cancers and for the risk-stratification of HPV infections. HPV sign Genotyping Test (QIAGEN) is a new pyrosequencing assay for the detection and genotyping of HPV. The sensitivity and comparative performance of HPV sign was determined using a sample panel derived from histologically confirmed cervical lesions (cervical intraepithelial neoplasia 2 or worse) and oropharyngeal squamous cell carcinomas. Comparative analysis showed that 80% of cervical intraepithelial neoplasia 2+ and 81% of oropharyngeal squamous cell carcinomas were HPV-positive by HPV sign compared to 100% of the cervical intraepithelial neoplasia 2+ and 81% of oropharyngeal squamous cell carcinomas by the digene HPV Genotyping RH Test (RH), and INNO-LiPA HPV Genotyping Extra assay, respectively. Fewer genotypes were detected overall by HPV sign than via the relevant comparator assays (10 vs 21 for cervical intraepithelial neoplasia 2+; 4 vs 9 for oropharyngeal squamous cell carcinomas) and also fewer multiple infections (9 vs 28 for cervical intraepithelial neoplasia 2+; 0 vs 4 for oropharyngeal squamous cell carcinomas). HPV sign results were more compatible with the comparator assay for the oropharyngeal squamous cell carcinoma samples (100%) than for the cervical samples (73%). These results suggest that HPV sign in its current form is suited to samples that harbour multiple infection.