The study was conducted to standardize a protocol for Agrobacterium-mediated genetic transformation of buffel grass (Cenchrus ciliaris L.). Embryogenic calli, produced from one-year-old mature seeds of buffel grass, were used as target cells for Agrobacterium-mediated transformation. A. tumefaciens strain LBA4404, harbouring pCAMBIA-1301 or pCAMBIA-2301, was used for co-cultivation with embryogenic calli from three genotypes (IG-3108, IG-9757 and IG-97101). Co-culturing of calli with Agrobacterium for 30 minutes, followed by co-cultivation with 0.1 mM acetosyringone for 3 days was found to be optimum for maximum transformation efficiency. Presence of acetosyringone during co-cultivation was found to be necessary for transformation. Transient GUS (-glucuronidase) gene expression was used to monitor T-DNA delivery into the target cells. Significant genotypic variations in response to transformation were observed among the tested genotypes. A very high frequency (63.3%) of GUS gene expression was obtained following Agrobacterium-mediated gene transfer into embryogenic calli. The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of buffel grass with genes of agronomic importance.