The intestinal microbial community is playing an important role in health and production performance of chickens. To understand the effect on the intestinal microflora induced by various feeding strategies, feed additives, infections, and intestinal disorders, it is important to have methods for quantifying potentially important bacteria in the intestine. We describe a real-time quantitative assay for detection and quantification of a whole group of Faecalibacterium prausnitzii and Subdoligranulum variabile-like bacteria, which has recently been found to dominate in the cecum of broiler chickens. The F. prausnitzii-S. variabile-like bacteria were quantified using a multiprobe assay in which one primer set was used to amplify DNA from all bacteria, whereas 2 probes detected all bacteria and the group of F. prausnitzii-S. variabile-like bacteria, respectively. The multiprobe assay accurately quantified the percentage of this group in a sample if it constituted more than approximately 5% of the total bacterial community. If the fraction of F. prausnitzii-S. variabile-like bacteria was lower than 5%, a duplex assay was applied in which the total bacteria were amplified in one tube and the F. prausnitzii-S. variabile-like group of bacteria was amplified in another tube using a specific forward primer. The F. prausnitzii-S. variabile-like group of bacteria was quantified in the cecum and ileum of conventional and organic raised chickens, in chickens kept in an isolator from 1 d of age, and in hatcher material. Quantification of this group of F. prausnitzii-S. variabile-like bacteria has not been performed before by real-time PCR, but results confirm previous results obtained by cloning and sequencing showing that the F. prausnitzii-S. variabile-like group of bacteria constitutes a major fraction of the cecal bacterial community in chickens. Furthermore, results indicate that the poultry farm environment plays a role in recruitment and development of these bacteria in the intestinal microflora.