In this study, Streptococcus gordonii-specific quantitative real-time polymerase chain reaction (qPCR) primers, RTSgo-F2/RTSgo-R2, were developed based on the nucleotide sequences of RNA polymerase β-subunit gene (rpoB). The specificity of the RTSgo-F2/RTSgo-R2 primers was assessed by conventional PCR on 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. PCR products were amplified from the genomic DNAs of only S. gordonii strains. The qPCR primers were able to detect as little as 40 fg of S. gordonii genomic DNA at a cycle threshold value of 33. These findings suggest that these qPCR primers detect S. gordonii with high specificity and sensitivity.