Japanese barberry (Berberis thunbergii DC.) is a popular ornamental shrub used in garden and urban landscaping. Currently there are over 60 B. thunbergii cultivars in the market. To better distinguish its cultivars, we used the amplified fragment length polymorphism (AFLP) technique to develop DNA marker profiles for 59 cultivars and hybrids. These markers were used to authenticate the trueness-to-name of B. thunbergii cultivars in production and in the market, control for intracultivar genetic variants, and develop a molecular key to identify cultivars approved for importation in Canada. Polymorphic markers from seven primer combinations were able to clearly differentiate 57 of 59 cultivars evaluated. Two cultivars, Aurea and Aurea Nana, could not be differentiated because they had identical marker profiles. Among the 274 plants tested, 263 were confirmed to be true-to-name and correctly labeled, whereas 11 plants could not be confirmed true-to-name. Seven of the 20 cultivars evaluated exhibited detectable intracultivar genetic variation. ‘Crimson Pygmy’ had the highest number of plants exhibiting genetic variability. Overall, nursery producers and retailers do not appear to be mixing or mislabeling cultivars. A molecular key developed from a subset of 25 markers was able to accurately identify and differentiate the 11 B. thunbergii cultivars approved for importation in Canada. This key could be used in a cultivar verification program to facilitate international trade of B. thunbergii cultivars where wheat rust is a concern.