Osmopriming has a positive effect on the enhancement of seeds germination and seedlings growth, especially under stress conditions. This study investigated the effects of osmopriming with polyethylene glycol on alfalfa (Medicago sativa L.) seeds germination and seedlings antioxidant responses under drought stress. Seeds of five Moroccan alfalfa populations and an American Moapa variety were used to investigate the effect of osmopriming on seeds germination, seedlings growth, activities of antioxidant enzymes and membrane stability under two water deficit levels (-0.45 and -0.75 MPa). Seeds were primed with polyethylene glycol (PEG6000) (-0.6 MPa) for 24 h at 25 °C. The results showed that treated seeds presented higher germination rate and growth of 8 d-old seedlings than untreated ones. Particularly, osmoprimed seeds of ’Adis-Tata’ (Ad) and ’Riche’ (Rc) populations presented the highest final germination percentages of 90.8% and 64%, respectively, and seedlings shoot and root lengths under both levels of water deficit. The priming treatment enhanced the activity of peroxidase (PO) and catalase (CAT) and reduced the malonyldialdehyde (MDA) content and the electrolyte leakage under water deficit. Generally, the success of germination was positively correlated to PO and CAT activities and the degree of membrane stability in drought tolerant populations. However, the positive effect of the osmopriming technique on alfalfa drought tolerance remains limited in some tested populations, and severe water stress could inhibit germination and cause damages of alfalfa seedlings.