Background: Human Papillomavirus (HPV) is the most common sexually transmitted infection and it is a well-established cause of cervical cancer. We conducted this study to ascertain the presence of HPV-16 in women ≥ 30 years with normal cytology, in order to optimise and up-date preventive interventions against HPV infection and cancer. Methods: It was a prospective study conducted over a six month period in 2008. Women were randomly selected from the general population of different municipalities from Havana. A total of 81 women aged 30 years or older without cytological abnormalities were included. A standardized protocol was used for cervical specimen collection and mucosal HPV testing was by MY09/11 polymerase chain reaction (PCR) with nucleotide sequencing. HPV-16 genotyping was performed by Immunocytochemistry and quantitative real-time PCR. Risk factors associated with HPV infections were also investigated. Results: The overall prevalence of high-risk HPV in women with cervical cytology negative for intraepithelial lesions or malignancy (NILM) was 53.1% (43/81) and of HPV-16 was 45.7% (37/81). The estimate of cumulative proportions of women free from HPV infection was 46.9% (38/81). A low educational level was significantly associated with HPV-positivity (p 0.038). Conclusions: Detection of oncogenic HPV genotypes in women ≥ 30 NILM in cervical screening test can be a part of a screening program policy to reduce the burden of HPV-associated cervical cancer. These analyses validate the 2006 American Society of Colposcopy and Cervical Pathology guidelines for HPV-16 genotyping, which recommend referral to colposcopy of HPV-16+ women with negative cytology.