In the present study, we have developed a high-frequency plant regeneration system for Italian ryegrass via callus culture using mature seeds as explants. Optimal embryogenic callus induction was found to occur in MS medium containing 5 ㎎ 1?¹ 2,4-D, 0.5 ㎎ 1?¹ BA, 500 ㎎ 1?¹ L-proline, 1 g 1?¹ casein hydrolysate, 30 g 1?¹ sucrose, 7 ㎎ 1?¹ AgNO₃, 2 ㎎ 1?¹ CuSO₄ and solidified with 3 g 1?¹ Gelrite. The highest regeneration rate was obtained in MS medium containing 1 ㎎ 1?¹ 2,4-D, 5 ㎎ 1?¹ BA, 500 ㎎ 1?¹ L-proline, 1 g 1?¹ casein hydrolysate, 1 ㎎ 1?¹ thiamine-HCl, 30 g 1?¹ sucrose, 7 ㎎ 1?¹ AgNO₃, 2 ㎎ 1?¹ CuSO₄ and solidified with 3 g 1?¹ Gelrite. By using the most effective treatment determined for each parameter, the highest rates of embryogenic callus formation (48.9%) and regeneration (47.6%) were obtained with the Hwasan 101 cultivar. The overall plant regeneration rates of the examined cultivars ranged from 7.5% to 23.2%. Thus, optimization of regeneration frequency using mature seeds as explant material may offer a simple and efficient protocol for Italian ryegrass that may improve molecular breeding of this species.