Background: Characterizing estradiol among women with HIV may have implications for breast cancer and cardiovascular disease risk but has not been adequately explored. We quantified differences in total (E2), free (FE2) estradiol, and sex hormone binding globulin (SHBG) by HIV and viral suppression status. Methods: Women from a substudy (2003-2006) within the Women's Interagency HIV Study (IRB approved at each participating site) were included if they reported: a period in the last six months, were not pregnant/breastfeeding, no oophorectomy, and no exogenous hormone use in the prior year. Serum was collected on days 2-4 of the menstrual cycle. We assessed differences in biomarkers at 25th, 50th, and 75th percentiles by HIV and viral suppression status using weighted quantile regression. Results: Among 643 women (68% with HIV) median age was 37 years. All E2 percentiles were significantly (p < 0.05) lower in women with suppressed viral load versus women without HIV (4-10 pg/mL). The 25th and 50th percentile of E2 were 4-5 pg/mL lower in women with unsuppressed viral load compared to women without HIV (p < 0.05). The 25th and 50th percentile of SHBG was significantly higher in women with unsuppressed viral load compared to women without HIV (10 and 12 nmol/L, respectively). There were no consistent differences in estradiol or SHBG by suppression status. Conclusions: There were no differences in FE2 but significantly lower E2 and higher SHBG among women with HIV versus without HIV. Further research is merited in a large contemporary sample to clarify the clinical implications of these findings.