BACKGROUND: Cervical cancer screening guidelines for women aged ≥30 years allow for co-testing or primary cytology testing. Our objective was to determine the test characteristics and costs associated with Cytology, HPV and Co-testing screening strategies. MAIN METHODS: Retrospective cohort study of women undergoing cervical cancer screening with both cytology and HPV (Hybrid Capture 2) testing from 2004 to 2010 in an integrated health system. The electronic health record was used to identify women aged ≥30 years who had cotesting. Unsatisfactory or unavailable test results and incorrectly ordered tests were excluded. The main outcome was biopsy-proven cervical intraepithelial neoplasia grade 3 or higher (CIN3+). KEY RESULTS: The final cohort consisted of 99,549 women. Subjects were mostly white (78.4 %), married (70.7 %), never smokers (61.3 %) and with private insurance (86.1 %). Overall, 5121 (5.1 %) tested positive for HPV and 6115 (6.1 %) had cytology ≥ ASCUS; 1681 had both and underwent colposcopy and 310 (0.3 %) had CIN3+. Sensitivity for CIN3+ was 91.9 % for Primary Cytology, 99.4 % for Co-testing, and 94.8 % for Primary HPV; specificity was 97.3 % for Co-testing and Primary Cytology and 97.9 % for Primary HPV. Over a 3-year screening interval, Primary HPV detected more cases of CIN3+ and was less expensive than Primary Cytology. Co-testing detected 14 more cases of CIN3+ than Primary HPV, but required an additional 100,277 cytology tests and 566 colposcopies at an added cost of $2.38 million, or $170,096 per additional case detected. CONCLUSIONS: Primary HPV was more effective and less expensive than Primary Cytology. Primary HPV screening appears to represent a cost-effective alternative to Co-testing.