Three S genome specific sequences were isolated from Aegilops sect. sitopsis species using different experimental approaches. Two clones, UTV86 and UTV39, were isolated from a partial genomic library obtained from DNA of Aegilops sharonensis, whereas a third clone, UTV5, was isolated from Aegilops speltoides. The three clones were characterized by sequencing, analysis of methylation, and sequence organization and abundance in some Aegilops and Triticum species. The clones UTV39 and UTV5 belong to the same family of tandem repeated sequences and showed high homology with a sequence already present in nucleotide databases. The UTV86 clone from Ae. sharonensis corresponded to an interspersed low frequency repeated sequence and did not show any significant homology with reported sequences. Southern hybridization experiments, using the cloned sequences as probes, detected polymorphism in the restriction patterns of all the five Aegilops species in section sitopsis. Aegilops speltoides showed the most divergent hybridization pattern. A close relationship was detected between the S genome of Ae. speltoides and the G genome of the wild Triticum timopheevii. In situ hybridization revealed a telomeric and (or) subtelomeric location of the sequences UTV39 and UTV5.