We screened regenerable genotypes from 12 cultivars of Italian ryegrass (Lolium multiflorum Lam.) through tissue culture of mature seeds, shoot tips and root tips. Although three morphological types of calli, friable, watery, and compact with fine root hair were observed, green shoots regenerated only from the friable calli. The highest frequency of the formation of regenerable callus from mature seeds was 1.4% in the diploid cultivar Waseaoba and 1.1% in the tetraploid cultivar Meritra. The plants regenerated from mature seed-derived calli were maintained and propagated aseptically in vitro, and the method of regenerating plantlets via callus culture was established by using their shoot tips. Sixty-three seeds of Waseaoba and 44 seeds of Meritra were aseptically sown, each seedling (genotype) was propagated in vitro, and the shoot tip from each genotype was subjected to tissue culture. More than 22% of these genotypes formed regenerable calli. From these results, we conclude that the shoot tip of the in vitro-preserved plantlet is useful for producing the regenerable calli routinely. The information obtained in the present study is useful for establishing a reliable transformation system for Italian ryegrass.