Evaluation of molecular Human Papillomavirus (HPV) testing into UK Cervical Screening Programmes is underway. In South Wales the current HPV prevalence in women attending routine screening is 13.5% with 76.3% HR HPV positive in cases with reported dyskaryotic cervical cytology [Hibbitts S, Jones J, Powell N, Dallimore N, McRea J, Beer H, et al. Human papillomavirus prevalence in women attending routine cervical screening in South Wales, UK: a cross-sectional study. Br J Cancer 2008;99(December (11)):1929–33].The aim of this study was to re-analyse the 23.7% cases with reported dyskaryotic cytology that were HR HPV negative (n=52 out of 219 in a population of 10,000).Three procedures were performed: (i) GP5+/GP6+ PCR-EIA repeat on original DNA extracts; (ii) DNA extraction and GP5+/GP6+ HPV PCR-EIA; (iii) DNA extraction and HPV typing using Greiner Bio-One™ PapilloCheck DNA microarray.51 out of 52 samples were re-analysed. Direct repeat HPV PCR-EIA identified 24% (n=12/51) of samples positive for HR HPV. Re-extracted DNA and PCR-EIA increased detection to 41.2% (n=21/51) and PapilloCheck detected 78.4% (n=40/51). HR HPV detection by PapilloCheck was significantly higher compared with the other methods of re-analysis.Eleven samples were persistently HR HPV negative but 4 tested positive for low risk HPV.This study identifies that up to 78% of samples with dyskaryotic cervical cytology that test negative for HPV can be found to be HPV positive on re-analysis. The reliance on a single negative HPV test result could lead to missed HPV related disease in a subset of patients, the number dependant on which HPV test is performed. The clinical significance of a false negative HPV result depends on the screening interval and how HPV testing is incorporated into screening.