We describe the use of capillary gel electrophoresis (CGE) based on rfbS allele-specific polymerase chain reaction (PCR) analysis to simultaneously detect Salmonella pullorum (S.pullorum) and Salmonella gallinarum (S.gallinarum), which are the major bacterial pathogens in poultry and humans.rfbS allele-specific PCR was used to concurrently amplify two specific DNA fragments with lengths of 147-bp and 187-bp at an annealing temperature of 54±1℃C and an MgC12 concentration of 2.8-5.6 mM for the simultaneous detection of both S.pullorum and S.gallinarum.Under an electric field of 333.3 V/cm and with a sieving matrix of 1.0% poly(ethyleneoxide) (Mr 600 000), the amplified PCR products were analyzed within 6 min by the CGE separation.The potential provided by translating this CGE assay to the microchip format using programmed field strength gradients (PFSG) was also demonstrated.In the microchip gel electrophoresis with PFSG, both of the Salmonella analyses were completed within 30 s, without decreasing the resolution efficiency.The rfbS allele-specific PCR-microchip gel electrophoresis with the PFSG technique may prove to be a new tool for the simultaneous detection of both S.pullorum and S.gallinarum, due to its ultra-speed and high efficiency.