Abstract Body: Obesity is a leading risk factor for hypertension and cardiovascular disease. Recent evidence indicates that obesity abolishes the protection typically conferred by female sex and predisposes young, premenopausal women to vascular dysfunction and hypertension. Work from our laboratory identified that obesity induces hypertension in females via activation of the leptin-aldosterone-mineralocorticoid receptor (MR) axis. However, the origin of this sex-specific mechanism remains unknown. Therefore, we tested the contribution of both sex hormones and chromosomes, hypothesizing that sex chromosomes may underlie this sexual dimorphism. Studies in obese female agouti yellow mice with preserved (sham) or depleted (ovariectomy (OVX)) sex hormones indicated that absence of sex hormones did not alter blood pressure (BP) measured via radiotelemetry, aldosterone levels, or adrenal aldosterone synthase (CYP11B2) expression. Vascular function measured via wire myography indicated OVX impaired endothelial relaxation with no further alterations to vascular function. Leptin receptor blockade decreased BP in both sham and OVX mice and restored endothelial-dependent relaxation, indicating that the absence of sex hormones does not alter the mechanism of hypertension. Since the standard chow diet used contains phytoestrogens, we tested whether dietary phytoestrogens influenced the studies investigating sex hormone contribution to BP. Sham and OVX female C57BL/6 mice were fed either a chow diet containing phytoestrogens (Teklad 2018) or a diet devoid of phytoestrogens (AIN-76A, Teklad). Neither OVX nor diet altered systolic BP, vascular function, or plasma aldosterone levels, ruling out a role for phytoestrogens in the lack of differences in OVX agouti mice. To further test the role of sex hormones, primary human adrenal and HAC15 cells were treated with estrogen (E2), progesterone (P4), E2+P4, or dihydrotestosterone, which induced no alterations in CYP11B2 expression, supporting no role for sex hormones in aldosterone production. Therefore, to test the role of sex chromosomes, we used the four-core genotype (FCG) mouse model. BP was recorded in OVX XX and XY females. OVX XX females displayed higher baseline BP and plasma aldosterone than XY females. Collectively, these findings support that female sex chromosomes, not hormones, potentially mediate aldosterone production and are likely the origin of the sex-specific mechanism underlying obesity-associated hypertension.