Impaired venous drainage of the lower extremities determines a cascade of pathologic events leading to chronic venous disease (CVD). It is believed that the one cause of CVD is red blood cell extravasation and local iron overload that could generate free radicals and iron-dependent inflammation. The aim of this study was to investigate the relationship between: the intracellular iron deposits in varicose veins and tissue oxidative state measured by: the Proton Induced X-ray Emission Spectroscopy (Fe(PIXE)), (tSOD), (tGPx), (tTBARs) and (boxDNA). Patients with diagnosed CVD were qualified for surgical procedure. Entire trunk of the great saphenous vein (GSV) was extracted. Part located near medial ankle was considered competent (C) in duplex ultrasonography (USG) examination. The incompetent (I) part was extracted from GSV where USG showed incompetent valves and massive venous reflux. The difference between local tFe(PIXE), tTBARS, boxDNA, tGPx, tSOD in incompetent and competent part of vein tissue was statistically significant. Intima/media ratio directly correlated with Fe(PIXE) C/I concentration. Iron deposition in competent vs incompetent part of vein was also related to the oxidative stress parameters (boxDNA). The findings from this pilot study suggest that Fe(PIXE) measurement may be useful for explaining the progression of chronic venous disease.