Persistent infection with high-risk human papillomavirus (HR-HPV) is a major contributor to the development of high-grade cervical lesions and invasive cervical cancer. The expression of HR-HPV E6/E7 oncoproteins is a key factor in the progression from preinvasive lesions to cervical cancer, making the detection of E6/E7 mRNA a valuable marker of disease progression. This prospective study evaluated human papillomavirus (HPV) E6/E7 mRNA expression levels and genotype distribution in women referred for colposcopy and biopsy in Yunnan Province, China. Out of 106,245 women undergoing routine checkups, 676 met the inclusion criteria for further analysis. Histological examinations revealed a total of 266 cases of cervicitis, 59 cases of cervical intraepithelial neoplasia (CIN1), 151 cases of CIN2, 84 cases of CIN3, 87 cases of invasive cervical cancer and 29 cases of other conditions, including vaginal intraepithelial neoplasia and warts. The HPV mRNA test demonstrated positivity rates of 47.7% for cervicitis, 52.5% for CIN1, 84.1% for CIN2, 85.7% for CIN3 and 93.1% for invasive cervical cancer. The sensitivity and specificity values for the HPV mRNA test were as follows: for CIN2+, sensitivity was 87.0% and specificity 50.3%; for CIN3+, sensitivity was 89.5% and specificity 40.0%; and for invasive cervical cancer, sensitivity was 93.1% with a specificity of 36.3%. The corresponding positive predictive values were 61.4% for CIN2+, 33.6% for CIN3+ and 17.8% for invasive cervical cancer, while the negative predictive values were 80.9%, 91.8% and 97.3%, respectively. The most common HR-HPV genotypes identified were HPV16 (143 cases), HPV18 (116 cases), HPV52 (78 cases) and HPV58 (69 cases). Age-specific analysis revealed HR-HPV prevalence rates of 56.4% in women aged 18–25, 69.3% in those aged 26–35, 65.4% in the 36–45 age group, 54.2% in women aged 46–59 and 67.9% in those over 60. Remarkably, HPV16, HPV18 and HPV52 were consistently the most prevalent high-risk genotypes across all age groups. These findings highlight the significant burden of HR-HPV infection in Yunnan Province and emphasise the importance of incorporating HPV E6/E7 mRNA testing into cervical cancer screening programs. Given the predominance of HPV 16, 18 and 52, future HPV vaccine formulations should prioritise these genotypes to enhance cervical cancer prevention among Chinese women.