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Abstract Background The new nine-valent vaccine against human papillomavirus (HPV) includes the four HPV genotypes (6, 11, 16, and 18) that are targeted by the older quadrivalent HPV vaccine, plus five additional oncogenic types (31, 33, 45, 52, and 58) remain significantly associated with high grade lesions. We aimed to determine the prevalence of high-risk HPV genotypes in unvaccinated subjects and the association of these genotypes with the incidence of high-grade lesions. We also assessed which, if either, of these two HPV vaccines could have prevented these cases. Methods This cross-sectional study, conducted from 4 January 2010 to 30 December 2011, was composed of 595 women attending the Hospital General Universitario de Elche (Spain) gynaecology department who were positively screened for opportunistic cervical cancer by pap smears and HPV detection during a routine gynaecological health check. The pap smear results were classified using the Bethesda system. HPV genotyping was performed with the Linear Array HPV genotyping test, and viruses were classified by the International Agency for Research on Cancer assessment of HPV carcinogenicity. Odds ratios (ORs) with their 95% confidence intervals (95% CI) were estimated by logistic regression, adjusting for age and immigrant status. The prevented fraction among those exposed (PFe-adjusted) was determined as a measure of impact. Results At least one of the additional five high-risk HPV genotypes present in the nine-valent HPV vaccine was detected in 20.5% of subjects. After excluding women with genotype 16 and/or 18 co-infection, high-risk genotypes (31, 33, 45, 52, and 58) were associated with a higher risk of intraepithelial lesion or malignancy: adjusted OR = 3.51 (95% CI, 1.29–9.56), PFe-adjusted = 0.72 (95% CI, 0.22–0.90). Genotypes that are still non-vaccine-targeted were detected in 17.98% of the women, but these were not significantly associated with high-grade lesions. Conclusion The greater protection of the nine-valent HPV vaccine is likely to have a positive impact because, in the absence of genotype 16 or 18 infection, these five genotypes on their own remained significantly associated with high-grade lesions.