Abstarct: Canola is one of the most important oilseed crops in the world. Despite the production of transgenic canola plants, transformation efficiency is low. In order to investigate the effects of Agrobacterium strains and ultrasonic waves on frequency of the gene transformation in canola, experiment were carried out baxsed on completely randomized design with 3 and 5 replications, respectively, using cotyledon explants in canola (cultivar Okapi) Agrobacterium strains belong to LBA4404, EHA101 and GV3850, containing pBI121 plasmid carrying the gus and nptII genes , were evaluated in experiments. Results of statistical analysis showed significant difference among Agrobacterium strains used in this study and the highest transformation frequency achieved in strain of GV3850 (3%) and lowest transformation frequency was belonging to strain LBA4404 (1%). In ultrasonic waves experiments Agrobacterium strain EHA101 was used. cotyledons explants exposed to different ultrasonic waves treatments (2, 5, 10 and 20 seconds) before they were co-cultured with Agrobacterium. Results of statistical analysis showed significant difference among ultrasonic treatments. The highest transformation frequency achieved in treatment of 20 seconds (10%) and lowest transformation frequency was belong to control (2%). PCR reaction using regenerated plants on selection medium, with specific primers of gus and nptII genes, In both experiments confirmed the amplification of fragments 521bp for gus and 1341bp for nptII gene.