The development of a protocol for high efficiency of embryogenic callus separation, maintenance and plant regeneration from the seeds of zoysiagrass cv. Zenith was studied. Embryogenic callus ratio is absolutely determined by genotype, but by adding high concentration of copper into medium, changing light condition and maintaining callus on initial induction medium for 8∼10 weeks, embryogenic callus can be easily distinguished and its growth can be promoted. There were significant differences among selected callus lines (each from one seed) according to their growth rates and regeneration percentages. Callus pre-treatment with activated charcoal inhibited callus growth, increased the level of precocious germination during culture and promoted shoot cluster formation after transfer to regeneration medium. For long-term callus maintenance, N6AA medium was better than MS medium, because the former inhibited non-embryogenic callus formation and kept vigor of embryogenic callus. The best callus lines Z-(5) has been successfully used for transformation and somaclonal variation selection in our laboratory.