There is little information about HIV awareness or condom use among female sex workers (FSWs) in Afghanistan. The purpose of this cross-sectional study was to assess HIV awareness, knowledge, and condom use among FSWs in three Afghan cities. FSWs residing in Jalalabad, Kabul, and Mazar-i-Sharif were recruited through outreach programs and completed an interviewer-administered questionnaire and rapid tests for hepatitis B surface antigen, HIV, syphilis, and hepatitis C virus. Logistic regression identified factors associated with HIV awareness, comprehensive HIV knowledge (knowledge that HIV cannot be detected by sight, that condoms prevent HIV, and rejection of local misconceptions about HIV transmission), and consistent condom use (use with every sex act) with clients in the last six months. Of 520 participants, 76.9% had no formal education and 37.7% lived outside Afghanistan in the last five years. Nearly half (44.2%) were aware of HIV but, of these, only 17.4% (N = 40) had comprehensive HIV knowledge. There were significant differences by site; FSWs in Jalalabad were more likely to be aware of HIV but FSWs in Kabul were more likely to have correct HIV knowledge and use condoms consistently with clients. Consistent client condom use was reported by 11.5% (N = 60) and was independently associated with having more clients per month (AOR = 1.99, 95% CI: 1.04-3.81). In conclusion, comprehensive HIV knowledge and consistent condom use with clients are low among Afghan FSWs in these cities. Efforts to reach this population should focus on relaying accurate information and expanding condom use with clients.