A stretch of nucleotides consisting of a conserved region and an adjacent upstream variable region in the 16S rDNA of members of domain Bacteria was identified as a suitable target site for developing a real-time PCR adjacent hybridization assay. A single universal fluorogenic cyanin 5-labelled probe, CY5 1046+, targeting the conserved region, and two FITC-labelled probes, Calo and Fervi, targeting the variable region were designed and synthesized for the identification and differentiation of the thermophilic anaerobes Caloramator and Fervidobacterium. The simultaneous hybridization of probes CY5 1046+ and Fervi to the 16S rDNA target sites of Fervidobacterium species during PCR amplification resulted in an increase in fluorescence emission that was monitored continuously in a LightCycler. The differences in the temperature of disassociation of the hybridization probes (T(m)) due to compositional variation in the nucleotide bases of the probe and target sequences enabled Fervidobacterium islandicum DSM 5733(T) to be differentiated from Fervidobacterium gondwanense ACM 5017(T) and Fervidobacterium nodosum ATCC 35602(T). The simultaneous hybridization of probes CY5 1046+ and Calo to the 16S rDNA target sequence of Caloramator indicus ACM 3982(T) during PCR amplification also resulted in an increase in fluorescence emission. However, as the target sequence of C. indicus ACM 3982(T) is identical to those of the other three Caloramator species, Caloramator fervidus ATCC 43204(T) (formerly Clostridium fervidus), Caloramator proteoclasticus DSM 10124(T) and Caloramator coolhaasii DSM 12679(T), further species discrimination on the basis of T(m) will not be possible, making probe Calo a useful genus-specific probe. The devised method was subsequently used to detect and identify Fervidobacterium and Caloramator isolates from our previously uncharacterized culture collection and from enrichment cultures. The assay is cheap and flexible, as only a battery of inexpensive FITC probes for differentiating other members of domain Bacteria needs to be synthesized and DNA templates prepared by a simple lyse-boil method, in addition to purified DNA, can also be used.