In order to find the best screening kanamycin concentration in the genetic transformation of mustard (Brassica juncea Coss.), the seedling cotyledons of mustard were placed on bud-induced media supplemented with different kanamycin concentrations. The bud differentiation of explants was totally inhibited when the kanamycin concentration was greater than 30 mg/L. The seeds of mustard were placed on germination media supplemented with different concentrations of kanamycin. All the seedlings were white when kanamycin concentration was higher than 200 mg/L. The leaves of mustard in field were smeared with the solutions including different concentrations of kanamycin. The treated leaves showed white when kanamycin concentration was over 200 mg/L. To study the segregation of the alien gene in transgenic mustard offspring, the transgenic mustard seeds and the leaves of the transgenic mustard offspring using npt-gene as assistant selection-marker were treated with 200 mg/L kanamycin, and the results were analyzed by chi-square test. The segregation ratio in the offspring of 4 transgenic lines with single copy of transgene agreed with a ratio of 3:1. The segregation ratio in offspring of the one transgenic line with double copies was agreed with a ratio of 3:1, and the segregation ratio in offspring of the other transgenic line with double copies was agreed with ratios of 3:1 and 15:1 simultaneously. It is indispensable to thoroughly study the insert of the double copies of transgenes in transgenic mustard. PCR technology was used to confirm the above detection methods. It is concluded that applying kanamycin to screen transgenic mustard offspring is feasible.