The effects petroleum hydrocarbons (PH) on the morphological and physiological responses of roots have not been well studied. This research evaluated the root hydraulic responses of Medicago sativa seedlings exposed to diesel. Seeds were germinated in test tubes with 1% water-agar contaminated with diesel (3000, 6000, 9000, 12000, 15000, 18000 and 21000μLL−1), and uncontaminated test tubes were used as controls (0μLL−1). Ten days-old roots were detached to determine root anatomy, hydraulic conductivity (Kh), specific hydraulic conductivity (Ks), percentage of loss conductivity (PLC), and leaf water potential (Ψlw). Root morphological changes were related to the loss of tetrarch arrangement; however, the number of metaxylem vessels increased in response to diesel. Increasing diesel concentrations resulted in diminished Kh and Ks values, as well as enhanced PLC values. Diesel contamination resulted in the accumulation of superoxide and hydrogen peroxide in root cells, as determined by confocal microscopy.