OBJECTIVES This study aimed to determine human papillomavirus (HPV) genotypes present in biopsy sections from young women of vaccine eligible age living in Victoria, Australia, with confirmed cervical intraepithelial neoplasia grade 3 (CIN3) or adenocarcinoma in situ (AIS) using laser capture microdissection (LCM). METHODS Histologically confirmed CIN3 or AIS positive biopsies from vaccine eligible women (born after 30th June 1981, n=169), between May 2011 and March 2013, were identified. CIN3 or AIS lesions were isolated from biopsy material using LCM, and the HPV genotypes present in whole tissue sections (WTS) as well as LCM-isolated lesion tissue were determined by a sensitive reverse hybridisation assay; RHA kit HPV SPF10-LiPA25, version 1 (Labo Bio-medical Products, Rijswijk, The Netherlands). RESULTS One hundred and sixty-eight cases were shown to be HPV positive (99%), of which 20 (12%) had more than one HPV genotype detected using WTS-PCR. Evaluation by LCM of individual biopsies with mixed infections showed 18 cases (90%) had only one HPV genotype associated with each CIN3 lesion. HPV 16 was the most common HPV type, found in 95/168 cases (57%). CONCLUSION LCM-PCR allowed us to confirm the presence of a single HPV genotype associated with each biologically separate CIN3 lesion, supporting the theory that only one virus type causes each independent CIN lesion. LCM will provide an important tool in assessing vaccine effectiveness in HPV vaccine programs.