Iron deficiency is an important environmental factor restricting plant productivity. Selecting tolerant genotypes is one of the possible ways to solve this problem. Many studies reported the effects of Fe deficiency on photosynthesis and anti-oxidative defense system. Yet, there is little information available on the use of these attributes as selective criteria. In the present study, we aim to determine some physiological and biochemical traits conferring Fe deficiency tolerance at leaf level in two lines of Medicago ciliaris. Our results showed that Fe deprivation had a lowering effect on photosynthesis (chlorophyll, photosynthetic electron transport activity and chlorophyll fluorescence) in both lines studied. However, the sensitive line TN8.7 was more affected. Hydrogen peroxide concentration was negatively correlated with the activities of antioxidant enzymes and with the concentration of some non-enzymatic antioxidant. The tolerant line TN11.11 was characterized by a more efficient antioxidant defense system in comparison with the sensitive line TN8.7. The main conclusion of this study is that photosynthesis and antioxidant defense system could be used as physiological and biochemical indicators of Fe deficiency tolerance in Medicago ciliaris plants.