Abstract: Simple, rapid, high efficient and specific, thermal asymmetric interlaced PCR(TAIL-PCR), a kind of chromosome walking based on PCR, was mainly used to isolate the flanking sequences of target DNA segments. The princi-ple, advantage, disadvantage and reaction system optimization of TAIL-PCR as well as some critical problem were summa-rized. In animal husbandry and veterinary medicine, TAIL-PCR was mainly used to isolate exogenous gene insertion sites and clone flanking sequences of the known genes in transgenic animals. Being limited by the complexity of genome DNA , there were so many limited factors preventing cloning target genes from genome, such as amplification of nonspecific products caused by mismatch of specific primers and arbitrary primers, and the difficulties in obtaining positive products even using multiple degenerate primers caused by abnormal template GC content and highly repeated sequences. So the adjustments should be made depend on actual situation. When the templates were complex, adapter could be added to the primers to improve the specificity based on the principle of LM-PCR; while when the template were simple or the sequence were clear, primers could be designed and reaction conditions could be set based on the characters of the templates (high GC content) or known template sequence, which made it easier to operate; and the suitable primers and reaction conditions could be chosen to get longer amplification products based on the principle of Suppression-PCR. Moreover , TAIL-PCR could be used with other technologies to facilitate its application in animal husbandry and veterinary.