BACKGROUND: Food insecurity is a well-established determinant of sub-optimal self-reported antiretroviral therapy (ART) adherence, but few studies have investigated this association using objective adherence measures. Concentration of ART in hair is an objective and validated measure of drug adherence and exposure. We examined the association of food insecurity with levels of ART concentrations in hair among women living with HIV (WLHIV) in the United States (US). METHODS: We analyzed longitudinal data collected semi-annually from 2013- 2015 from the Women's Interagency HIV Study, a multi-site prospective cohort study of WLHIV and HIV-negative controls. Our sample comprised 1,944 person-visits from 677 WLHIV. Food insecurity was measured using the US Household Food Security Survey Module. ART concentrations in hair were measured using high performance liquid chromatography with mass spectrometry detection for regimens including darunavir, atazanavir, raltegravir or dolutegravir. We conducted multiple three-level linear regressions that accounted for repeated measures and the ART medication(s) taken at each visit, adjusting for sociodemographic and clinical characteristics. RESULTS: At baseline, 59% of participants were virally suppressed and 45% reported food insecurity. In the base multivariable model, each 3-point increase in food insecurity was associated with 0.94-fold lower ART concentration in hair (95% CI: 0.89, 0.99). This effect remained unchanged after adjusting for self-reported adherence. CONCLUSIONS: Food insecurity was associated with lower ART concentrations in hair, suggesting that food insecurity may be associated with sub-optimal ART adherence and/or drug absorption. Interventions that aim to improve ART adherence among WLHIV should consider and address the role of food insecurity.