Human papillomavirus (HPV) infection has been implicated as a causative factor of cervical cancer. This study aimed to examine HPV genotype distribution and prevalence among women in Hunan province, mid-south China.Cervical samples were collected from 3640 women for cervical cancer screening. Participants were screened by cytology, and HPV genotyping was performed by DNA chip.Of the 3640 cases, 22.6% (822/3640) were HPV DNA positive, of whom 19.0% (156/822) had multiple infections and 20.0% (726/3640) were confirmed to have high-risk HPV infection. The most common HPV genotype was HPV-16, followed by -52, -58, -18, -6 and -39. Cytological examination showed that the HPV positive rate was 59.3% (80/135) in women with atypical squamous cells of undetermined significance, 66.1% (111/168) in women with low-grade squamous intra-epithelial lesions, 72.6% (106/146) in women with high-grade squamous intra-epithelial lesions, and 87.2% (68/78) in women with invasive cervical cancer, all of which were significantly higher than the rate in women with normal squamous cells (14.3%, 451/3115).This is the first study to report the prevalence and genotype distribution of HPV infection among women in Hunan province, China. The findings provide important guidance for a vaccination programme in this region aimed at immunizing women before they are infected with HPV.