Objective: To evaluate the effectiveness of five methods including the ThinPrep cytological test (TCT), liquid-based cytology, the human papillomavirus (HPV) test, detection of the TERC and C-MYC genes and visual inspection with acetic acid/Lugol's iodine (VIA/VILI) for opportunistic cervical cancer screening, and to explore whether genomic amplification of the human telomerase gene and C-MYC in liquid-based cytological specimens can be used as a method for opportunistic cervical cancer screening. Methods: Data were collected prospectively from 1,010 consecutive patients who visited the gynecology clinic and agreed to participate in opportunistic cervical cancer screening at our institution from November 2010 to July 2011. The five methods mentioned above were used for the screening in all cases. The histopathological diagnosis served as the gold standard for the evaluation. A comparison between the five screening methods for the diagnosis of high-grade cervical intraepithelial neoplasia (CIN II and III) was performed for their sensitivity, specificity, false-positive rate, false-negative rate, accuracy rate, positive likelihood ratio and negative likelihood ratio. A comprehensive comparison of the different combination programs for screening was performed according to the analysis of the receiver operating characteristic (ROC) curve area. The accuracy of the five screening methods for the diagnosis of high-grade CIN (CIN II and III) was compared in the different age groups. A joint model for the diagnosis using different combinations of the five methods was developed according to the analysis by the SAS 8.0 software. The model was used to evaluate the accuracy of the different combination programs for the diagnosis of high-grade CIN, and the results were confirmed by the histopathological examination. Results: The sensitivity and specificity of the single screen method (TCT, HPV test, detection of the TERC and C-MYC genes, and VIA/VILI method) for CIN II was 80.9, 70.2, 72.3, 76.6, and 72.3%, as well as 98.0, 95.1, 96.3, 96.3, and 90.4%, respectively. The sensitivity of the single screening method in four different age groups (25-34, 35-44, 45-54 and 55-66 years) was as follows: TCT, 64.3, 90.9 76.5, and 85.7%; HPV test, 78.6, 72.7, 60.0, and 71.4%; the TERC gene, 50.0, 90.9, 80.0, and 71.4%; the C-MYC gene, 50.0, 90.9, 80.0, and 100%; VIA/VILI, 85.7, 81.8, 66.7, and 42.9%. The specificity was: TCT, 98.9, 98.1, 98.8, and 95.2%; HPV test, 96.7, 95.1, 92.2, and 100%; the TERC gene, 95.0, 98.9, 94.0, and 95.2%; the C-MYC gene, 97.2, 97.3, 93.4, and 97.6%; VIA/VILI, 91.2, 90.5, 89.8, and 88.1%, respectively. In the joint model for the diagnosis using different combinations, we found Logit (P) = 5.757 - 4.055 × TCT - 3.724 × HPV. The sensitivity and specificity in the combination program with TCT (primary screening) and HPV testing (adjunct screening) were 78.7 and 99.5%, while in the combination with HPV (primary) and TCT (adjunct), they were 53.2 and 99.7%, respectively. However, in the cytology-HPV parallel test, they were 97.9 and 93.4%. The ROC analysis revealed that the cytology-HPV parallel test is superior to the combinations of either TCT (primary) and HPV (adjunct) or HPV (primary) and TCT (adjunct; AUC<sub>TCT-HPV</sub> parallel test = 0.956; AUC<sub>TCT</sub>/primary<sub>HPV</sub>/adjunct = 0.764). Conclusions: Opportunistic cervical cancer screening is a practical approach to improve the efficiency of cervical cancer screening. Although the accuracy of TCT is the highest of the five screening methods for the diagnosis of high-grade CIN, it is still subject to sample acquisition and the practitioner's skill and experience. Since the efficacy of VIA/VILI may vary in all ages, it is not recommended for menopausal and perimenopausal women.