BACKGROUND: Heterosexual contact is the most common mode of HIV transmission in India that is largely linked to sex work. We assessed the non-use of condoms in sex work and with regular sex partners by female sex workers (FSWs), and identified its associations that could assist in planning HIV prevention programmes. METHODS: Detailed documentation of various aspects of sex work, and sexual behaviour with regular sex partners, was done through confidential interviews for 6,648 FSWs in 13 districts in the Indian state of Andhra Pradesh. Multivariate analysis was done to understand condom non-use with clients. RESULTS: 5,010 (75.4%), 1,499 (22.5%), and 139 (2.1%) FSWs were street-, home-, and brothel-based, respectively. Of the total 6,648 FSWs, 6,165 (92.7%) had penetrative vaginal/anal sex with at least one client in the last 15 days, and of these 2,907 (47.2%; 95% CI 41.2-53.2%) reported non-use of condom with at least one of her last three clients. Lack of knowledge that HIV could be prevented (odds ratio 5.01; 95% CI 4.38-5.73), no access to free condoms (odds ratio 3.45; 95% CI 2.99-3.98), being street-based as compared with brothel-based (odds ratio 3.36; 95% CI 1.87-6.04), and no participation in FSW support groups (odds ratio 2.02; 95% CI 1.50-2.70) were the most significant predictors of condom non-use with clients. Other associations included lower social support, lower income, age >24 years, illiteracy, and living in medium-size urban or rural areas. Of the 2582 who had penetrative sex with regular sex partner within the last 7 days, 2428 (94%; 95% CI 92.1-95.9%) had not used condom at last sex, and 1032 (41.8%) had neither used condom consistently with clients nor with regular sex partner. CONCLUSION: About half the FSWs do not use condom consistently with their clients in this Indian state putting them at high risk of HIV infection. Non-brothel-based FSWs, who form the majority of sex workers in India, were at a significantly higher risk of HIV infection as compared with brothel-based FSWs. With their high vulnerability, the success of expansion of HIV prevention efforts will depend on achieving and sustaining an environment that enables HIV prevention with the non-brothel based FSWs.