Fluorescence in situ hybridization (FISH) provides an efficient system for cytogenetic analysis of wild relatives of wheat for individual chromosome identification, elucidation of homoeologous relationships, and for monitoring alien gene transfers into wheat. This study is aimed at developing cytogenetic markers for chromosome identification of wheat and Aegilops geniculata (2n = 4x = 28, (UUMMg)-U-g-M-g-M-g) using satellite DNAs obtained from flow-sorted chromosome 5M(g). FISH was performed to localize the satellite DNAs on chromosomes of wheat and selected Aegilops species. The FISH signals for satellite DNAs on chromosome 5M(g) were generally associated with constitutive heterochromatin regions corresponding to C-band-positive chromatin including telomeric, pericentromeric, centromeric, and interstitial regions of all the 14 chromosome pairs of Ae. geniculata. Most satellite DNAs also generated FISH signals on wheat chromosomes and provided diagnostic chromosome arm-specific cytogenetic markers that significantly improved chromosome identification in wheat. The newly identified satellite DNA CL36 produced localized M-g genome chromosome-specific FISH signals in Ae. geniculata and in the M genome of the putative diploid donor species Ae. comosa subsp. sub-ventricosa but not in Ae. comosa subsp. comosa, suggesting that the M-g genome of Ae. geniculata was probably derived from subsp. subventricosa.