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HIV prevalence is estimated to be 2.7% in South Sudan; however, little is known about the young country's epidemic. We conducted a respondent-driven sampling biobehavioral survey in Juba of female sex workers (FSW) aged ≥15 years who sold or exchanged sex in the last 6 months to learn more about this population. We enrolled 838 FSW from November 2015 to March 2016 and estimated HIV prevalence to be 37.8%. Prevalence of active syphilis was 7.3%. FSW were from South Sudan and most neighboring countries. Comprehensive knowledge of HIV was 11.1% and 64.2% of FSW had never spoken with an outreach worker. In multivariable analysis, HIV was associated with being from Uganda (aOR: 3.3, 95% CI: 1.7-6.1) or Kenya (aOR: 4.3, 95% CI: 1.5-13.0) versus from South Sudan. Our survey suggests that FSW may play a critical role in South Sudan's HIV epidemic and highlights the importance of tailoring services to the unique needs of FSW of all nationalities in Juba.