AbstractPloidy manipulation has been used in many crop improvement programs to develop plant species with wider adaptability and desirable traits. The objectives of this study were to create stable tetraploid centipedegrass (Eremochloa ophiuroides [Munro] Hack.) lines and evaluate them for beneficial traits. To generate polyploid lines, “TifBlair” (2n = 2x = 18) seeds were exposed to gamma radiation and callus was generated using tissue culture, exposed to glyphosate and regenerated into plantlets. After 5 years of continuous propagation, two lines, Hongliang Wang (HW)16 and HW123, were found to be tetraploid, and one line, HW61, was found to be a mixoploid using flow cytometry and chromosome counts. Nuclear DNA contents for diploid and tetraploid lines ranged from 1.97 to 2.10 and 4.14 to 4.30 pg 2C⁻¹, respectively. Ten centipedegrass lines were genotyped using seven simple sequence repeat markers and clustered by origin (derived from TifBlair or the University of Georgia breeding program). Six lines were evaluated for morphological and physiological traits under greenhouse conditions. Trait evaluation showed that tetraploid lines had larger stomata and leaf width and reduced stomatal density. For all other traits evaluated under greenhouse conditions, although significant differences were observed between lines, no consistent differences separated diploid and tetraploid lines. Between the tetraploid lines, HW16 had higher evapotranspiration, percentage green cover, and relative water content than HW123, whereas HW123 had a higher average clipping yield and root width than HW16. In conclusion, irradiation and tissue culture are valuable techniques for the generation of stable polyploid lines, and an increased ploidy level led to larger vegetative structures in centipedegrass.