Abstract:Abstract: Traditional method of screening homozygous transgenic plants is several generations self-pollenation, this is a time consuming work and low efficiency. We created a new method by flanking sequences amplification of T-DNA insertion site to screen homozygous plants. Firstly, we using modified adaptor ligation PCR method amplified flanking sequences of T-DNA in tomato transgenic plants. Then flanking sequences were comfirmed by PCR analysis in transgenic plants. BLAST results in SGN database showed that T-DNA integration site lost 40 base pair. Finally, we screened out homozygous lines in T1 transgenic generation using flanking sequence primers and border sequence primers, and verified the results in T2 generation.