The copies of outside gene and DNA structure of integration locus are important in high expression and avoidance of gene silence. Many research results showed that outside genes were inserted into the plant genome with recombinant type, and the integration was related to border T-DNA sequence in the course of Agrobaterium mediated transformation. SAR structure was also found in the integration location of transformants with direct DNA transformation method. The pollen-tube pathway transformation, one of direct DNA transformation method, was very successfully used in Bt transgenic cotton in China. But until now there has been no report about Bt gene integration. The aim of this research was to investigate flanking DNA structure in order to explain the mechanism of direct transformation method in the future. The structure of flanking DNA fragments of Bt integration in four different transgenic Bt cotton varieties including Simian-3, 161 resistant breeding line, 161 sensitive breeding line and Guokangmian-1 was analyzed with TAIL-PCR and nested PCR. The flanking DNA sequences of different self-crossed progenies from one plant are the same. In contrary, their DNA sequences are diverse for different breeding lines. Upstream flanking fragments in some transgenic cotton contained short transformation plasmid sequences, downstream flanking fragments in all the transgenic cotton varieties were composed of high percentage AT. The percentage of AT in Simian-3 transgenic Bt variety was as high as 92%. All the flanking fragments were of multi-copies and similar to repetitive sequences. No recognition sites of TOP enzyme were found in these fragments.