Following the recent outbreak of Shiga toxin-producing Escherichia coli (STEC) O104:H4 infection in Germany, the demand for fast detection of STEC has again increased. Various real-time PCR-based methods enabling detection of Shiga toxin genes (stx) have been developed and can be used for applications in food microbiology. The present study was conducted to evaluate the reliability of seven commercially available real-time PCR systems for detection of stx1 and stx2 subtypes. For this purpose, pure cultures of 18 STEC strains harboring all known stx1 and/or stx2 subtypes were tested. Only one of the seven real-time PCR systems detected all known stx1 and stx2 subtypes. Six systems failed to detect the stx2f subtype. One system missed stx2 subtypes reported in association with severe human disease. Because the presence of certain stx genes (subtypes) is considered an important indicator of STEC virulence, systems differentiating between the stx1 and stx2 gene groups provide added value. Reliable and fast detection of stx genes is of major importance for both diagnostic laboratories and the food industry.