The authors compared the clinical performance of DH3 human papillomavirus (HPV) assay, which detects 14 high-risk HPVs with 16/18 genotyping based on hybrid capture technique, and Hybrid Capture 2 (HC2) test for women undergoing cervical cancer screening. A total of 7, 263 residual cytology specimens from an adjudicated cohort with 3-year follow-up were tested by the DH3 assay and the HC2 test. Assay results were compared with each other and to histology review. The overall agreement between the DH3 assay and the HC2 test was 99.2% (κ = 0.938). At baseline, DH3 had the equal sensitivity to that of HC2 for cervical intraepithelial neoplasia (CIN) grade 2 or higher (CIN2+,  = 75) and CIN grade 3 or higher (CIN3+,  = 45), 98.67% and 97.78%, respectively. After 3 years of follow-up, the sensitivity for CIN2+ ( = 133) and CIN3+ ( = 74) were both similar between DH3 and HC2 (95.49% vs 94.74%, 95.95% vs 95.95%, respectively, all  > 0.05). The respective specificity for CIN2+ or CIN3+ did not differ between the two tests. A noninferiority test showed that both sensitivity and specificity of DH3 for CIN2+ and CIN3+ were noninferior to those of HC2 at baseline and after 3-year follow-up, respectively (all  < 0.001). When used in primary screening strategy, the DH3 assay would yield an immediate sensitivity of 92% for CIN2+. DH3 HPV performs equally to HC2 for the detection of high-grade lesions in cervical cancer screening and has a potential advantage in primary screening strategy due to HPV16/18 genotyping. The benefits of testing for high-risk human papillomavirus (hrHPV) in cervical cancer screening have already been demonstrated. Hybrid Capture 2 (HC2) is the best validated HPV assay and has been considered the gold standard for hrHPV testing. However, HC2 cannot discriminate HPV16 and 18 from the other hrHPV types, which greatly limited the application of HC2 in cervical cancer screening. The DH3 human papillomavirus (HPV) is a recently developed assay based on hybrid capture technique like to HC2, which can specifically identify HPV 16/18 on the basis of detecting the 13 hrHPV types targeted by HC2 as well as HPV66. This comparative study of the two assays for detection of hrHPV infection in residual cytology samples from cervical cancer screening setting reveals that DH3 HPV provides a perfect alternative to HC2 in detecting hrHPV infection and identifying cervical precancer, while allowing concurrent HPV 16/18 genotyping.