It is very useful to detect bar gene in transgenic and non-transgenic wheat by a method with convenience, swiftness, and reliability for the screening of stable transgenic lines, the development of marker-free transgenic plants, inspection of commercial wheat varieties and products. Three identification methods of leaf painting, plant spraying, and mature embryos in vitro culture with the herbicides corresponding to bar gene were compared and evaluated in this study. Results indicated that transgenic or non-transgenic wheat plants and seeds with or without bar gene could be distinguished well by the three methods mentioned above. Application of leaf painting with 0.1% Liberty for the confirmation of the herbicide transgenic plants was better than PCR analysis in accuracy. The optimal concentrations for plant spraying were 100ppm for Basta and 150ppm for Liberty, and the available amount of bialaphos in medium for the intro mature embryos is 5-8 mg L-1. However, the first two methods were influenced in some extent by environmental conditions such as temperature, light, and nutrition in practice, which often led to an unclear observation in symptom for detecting the bar plants. In comparison, mature embryo in vitro culture on the medium with herbicide was less dependant on environment, and showed some advantages including clear observation, easy manipulation, and short time expense. Therefore, mature embryo in vitro culture identification was strongly suggested to be used in the detection and inspection of bar gene in transgenic and commercial wheat.