A complete protocol of in vitro selection and greenhouse screening for glyphosate-tolerant variants in manilagrass (Zoysia matrella [L.] Merr) was established in this study. Newly subcultured calli of more than 5 years’ old were transferred to selection medium containing 2 mM glyphosate. After two rounds of selection, 220 calli survived out of 840 and were transferred to regeneration medium without glyphosate. Regenerated plantlets were then transferred to regeneration medium containing 0.5 mM glyphosate to select tolerant plantlets. After 1-month growth, there were plantlets remained green and new shoots formed beside or on discolored explants. These surviving organisms were then transferred to fresh regeneration medium for further growth. Fully developed plantlets were transferred to a green house and then subjected to greenhouse screening by foliar spraying with 0.05 % glyphosate solution. Six glyphosate-tolerant plantlets, TP1-TP6, were obtained and proliferated for determination of sod-tolerance using morphological and physiological measurements. Fourteen days after foliar application with 0.1 % glyphosate, only TP5 showed enhanced sod-tolerance. The dark green color index value of TP5 was significantly higher than CK2, demonstrating that TP5 suffered less injury from glyphosate than CK2. Different physiological characters were also observed in CK1, CK2 and TP5. Significantly higher chlorophyll a content and catalase activity were observed in TP5 than in CK1. Fourteen days after treatment (DAT), the ion leakage, proline content and ascorbate peroxidase activity of CK2 and TP5 increased significantly, but the ion leakage of TP5 was significantly lower than that of CK2. The guaiacol peroxidase activity of TP5 increased significantly 14 DAT, and was significantly higher than that of CK1 and CK2. No change in shikimate content was observed in CK2 or TP5 14 DAT.