Salmonellosis in poultry of Korea is a significant health problem, which causes substantial economic losses. The most common causative agents of chicken salmonellosis ar S. gallinarum and S. pullorum. Traditional methods used to detect Salmoenella spp. In chicken are tedious, time consuming and confer little guarantee of sensitivity and species specificity. Therefore, a rapid and sensitive method for the differentiation of Salmonella serogroup D was assessed. We first amplified the rfbS genes by PCR and characterized the amplified product by nucleotide sequence analysis. The homology of nucleotide sequence was 99.7% between S. gallinarum and S. pullorum rfbS genes. Further comparisons of the sequences of S. gallinarum, S. gallinarum fied strain, S. pullorum and S. typhi(GenBank Accession No.M29682) showed a homology of 98.3%. The predicted amino acid sequence homology was 97.1%, 97.1% and 97.5%, respectively. Based on this comparison of these nucleotide sequences, we found unique restriction enzyme sites within the rfbS genes of S. gallinarum and S. pullorum. Thus, the PCR products could be further digested with restriction enzymes TfiI and PleI for use in a restriction fragment length polymorphism (RELP) technique. This method can be applied in the differential diagnosis between S. gallinarum and S. pullorum.