Among women in the United States, non-Latina black women in the South have disproportionately high rates of new HIV infections but low use of pre-exposure prophylaxis (PrEP). Effective strategies to identify factors associated with PrEP eligibility could facilitate improved screening, offering, and uptake of PrEP among US women at risk of HIV. We applied 2014 CDC criteria for PrEP use to at-risk HIV-negative women enrolled in the Southern US sites (Atlanta, Chapel Hill, Birmingham/Jackson, Miami) of the Women's Interagency HIV Study from 2014 to 2015 to estimate PrEP eligibility and assess PrEP knowledge and acceptability. Factors associated with PrEP eligibility were assessed using multivariable models. Among 225 women, 72 (32%) were PrEP-eligible; the most common PrEP indicator was condomless sex. The majority of PrEP-eligible women (88%) reported willingness to consider PrEP. Only 24 (11%) PrEP-eligible women had previously heard of PrEP, and only 1 reported previous use. Education level less than high school adjusted odds ratio (aOR) 2.56; 95% confidence interval (CI): 1.22 to 5.37, history of sexual violence (aOR 4.52; 95% CI: 1.52 to 17.76), and medium to high self-perception of HIV risk (aOR 6.76; 95% CI: 3.26 to 14.05) were significantly associated with PrEP eligibility in adjusted models. Extremely low PrEP awareness and use despite a high proportion of eligibility and acceptability signify a critical need to enhance PrEP education and delivery for women in this region. Supplementing CDC eligibility criteria with questions about history of sexual violence and HIV risk self-assessment may enhance PrEP screening and uptake among US women.