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Mentha species are planted worldwide for extraction of their metabolites, including essential oils and phenolic acid compounds, which have therapeutic and commercial value. Mentha piperita L. and Mentha canadensis L. both belong to the menthol chemotype. M. piperita is widely used in North America, and M. canadensis is one of the most important mint species in Asia. Here, an improved Agrobacterium-mediated transformation method using internode segments instead of leaves as explants of M. piperita was reported. Histological examination showed that adventitious buds formed from cortical cells of internode. An efficient method for regeneration of M. canadensis was also established. Internode pieces were excised and cultured on a regeneration medium containing Murashige and Skoog (MS) salts plus vitamin B5, 5.0 mg L−1 TDZ, 0.2 mg L−1 IAA, and 25% coconut water. Explants were then moved to the basal MS medium without plant growth regulator after initiation of shoots and regeneration rates achieved 100%. According to different selection markers used in individual transformation experiments, 3 mg L−1 hygromycin B (antibiotic) or bialaphos (herbicide) was appropriate the concentration for the regeneration selection stage, while 5 mg L−1 hygromycin B or bialaphos could be used during the shoot elongation selection stage. An Agrobacterium-mediated genetic transformation protocol for M. canadensis was produced within 4 mo. Positive transgenic lines with gusA gene were verified by polymerase chain reaction (PCR) and β-glucuronidase histochemical staining. Using 500 to 600 internode pieces as explants could obtain 3 to 4 transgenic lines of M. canadensis. Our methods established here will facilitate the basic gene function research as well as genetic breeding and trait improvement of this important mint species. [ABSTRACT FROM AUTHOR]