Abstract: The effect of Agrobacterium mediated on transformation frequency of different genotypes of cotton was studied in this experiment. Huakang6 and 06S62, were selected as the materials, which were two different genotypes of upland cotton . By changing the different of concentration of Agrobacterium, duration of infection and negative pressure, the proper transformation condition was confirmed. The results showed that the suitable conditions of Agrobacterium mediated transformation of cotton was that Huakang6, the concentration of Agrobacterium of OD6000.7, duration of infection for 10min, negative pressure for 10min was better. 06S62, the concentration of Agrobacterium of OD6000.9, duration of infection for 10min, negative pressure for 10min was better. The transformation frequency of Huakang6 was higher than that of 06S62. Its peak transformation frequency was 8.81%. Using the genetic transformation system, bar was introduced into the two genotypes of cotton, and genetically modified herbicide-resistant plants were obtained.