AbstractA multiple‐primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or more sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure. This strategy is suited for selective detection and genotyping of relevant microorganisms and samples harboring different DNA targets such as multiple variant/infected samples as well as unspecific amplification products. This method is used here in a model system for detection and typing of high‐risk oncogenic human papilloma viruses (HPVs) in samples containing multiple infections/variants or unspecific amplification products. Type‐specific sequencing primers were designed for four of the most oncogenic (high‐risk) HPV types (HPV‐16, HPV‐18, HPV‐33, and HPV‐45). The primers were combined and added to a sample containing a mixture of one high‐risk (16, 18, 33, or 45) and one or two low‐risk types. The DNA samples were sequenced by the Pyrosequencing™ technology and the Sanger dideoxy sequencing method. Correct genotyping was achieved in all tested combinations. This multiple‐sequencing primer approach also improved the sequence data quality for samples containing unspecific amplification products. The new strategy is highly suitable for diagnostic typing of relevant species/genotypes of microorganisms.