Pullorum disease and Fowl typhoid are kind of poultry specific disease for poultry. The peculiar character of these poultry specific diseases is that it can be infected by transmitting vertically and horizontally, also it is hard to be discovered by clinical sign, and pathology or immunology. So, to develop the PCR method which distinguishes these two genetically similar diseases of separated the specific DNA fragment from each strain and use it for differential diagnosis by subtraction PCR method. Standard strain of S gallinarum and S pullorum, and field isolation strain were verified by biochemistry, It confirmed existence of plasmid by using the PFGE. Then, Isolated DNA from it and used it as materials for the experiment. After cutting genomic DNA of two strains by using Sau 3Al, It ligated primer to tester DNA for PCR amplification and separated specific DNA fragment bacteria with method of subtraction PCR. And, It confirmed that it is a piece of unique DNA in every bacteria using base sequence of separated DNA fragment. 1. The six specific DNA fragment were separated from the DNA of S gallinarum and S pullorum by the subtraction PCR method. 2. In the result of comparison after setting base sequence of each fragment, each separated base sequence of DNA fragment they did not correspond to each other 3. As the result of each DNA fragment is derived from the each strain of DNA, and there was no homology of genomic DNA level in mutual. 4. The fragment originated in plasmid and includes S pullorum did not separate. 5. In the result of searching base sequence in Genebank, it partially shows homology in Salmonella enterica, S typhimurium, S dublin, Escherichia coli, Shigella flexneri, Yersinia pestis, Klebsiella pneumoniae. 6. Primer design by S gallinarum DNA 2, 3 fragment used PCR, They are positive reaction in only S gallinarum at 276, 367 bp position.