Recombineering is a new developed genetic engineering technology in the past few years. A new recombineering system named pYM-Red was constructed by gap-repair, that is a technology called in vivo cloning. Linear PCR fragments that amplified from low copy plasmid pACYC184 were used as gene targeting vector. The length of subcloned DNA sequence including Red gene and a series regulatory sequences are about 6.7 kb. The biology function of Red gene in pYM-Red was tested by gene replacement (galk<>kan) of W3110 chromosome. Factors that effect recombination efficiency were precisely confirmed. When induced 10 min at 42 ℃ and using 300 ng linear DNA fragment as targeting molecules, the efficiency of pYM-Red mediated recombination can reach one positive recombination clone per four thousands electroporation survived cells, it is 5~6 folds higher than pBR322-Red and pKD46 recombination system.