Objective: Chronic renal disease and hypertension are strongly associated with vascular damage, endothelial dysfunction (ED) and increased vascular stiffness. We hypothesized that several novel, potentially more sensitive biomarkers of kidney damage may be differently related to vascular stiffness and reactivity in male and female patients with different severity of hypertension. Design and method: Urine levels of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), liver fatty-acid binding protein (L-FABP) and serum levels of Cystatin C (sCys) and serum creatinine (sCr) were measured by quantitative enzyme immunoassay in 92 hypertensive patients, 46 males (mean age 46,3 ± 13,4 years) and 46 females (mean age 55,2 ± 8,9 years). Renal function (GFR) was analyzed using MDRD and the CKD Epidemiology Collaboration (EPI) sCr equation and CKD-EPI sCys equation. Instrumental examination was performed after 5 days of discontinuation of antihypertensive medications including applanation tonometry (SphygmoCor, Artcor Medical) with the calculation of central aortic pressure, pulse wave velocity (PWV) and augmentation index (AI). Endothelial function was assessed by reactive hyperemia index (RHI) with EndoPAT device (Itamar Medicals). Results: Female patients were characterized by higher NGAL (16.6 +/− 12.8 versus 6.0 +/− 4.3 ng/ml, respectively; p = 0.0001) and lower sCys-estimated GFR (88.0 +/− 14.1 mL/min/1.73 m2 versus 95.6 +/− 19.1; p = 0.04), while there were no differences in sCr-estimated GFR, KIM-1, L-FABP and Cystatin C levels. There were also no differences in RHI and PWV levels between groups. In females novel biomarkers levels were associated with increased arterial stiffness (PVW > 10 m/s): sCys-estimated GFR (r = 0.351, p = 0.02), Cystatin C and L-FABP (r = 0.285, p = 0.06; r = 0.405, p = 0.01, respectively). In males patients Cystatin C and L-FABP levels were associated with lower RHI (r = −0,704, p = 0.005; r = −0.651, p = 0.03, respectively), KIM-1 levels were associated with increased PWV (r = 0.612, p = 0.06) only in patients with severe and resistant hypertension. Conclusions: Cystatin C and L-FABP seem to be associated with increased arterial stiffness in hypertensive females and vascular reactivity in severe, resistant to treatment hypertensive males. Arterial stiffness in males is linked mostly to KIM-1 levels.