Thirty-three populations belonging to the three Retama species, Retama monosperma, Retama raetam and Retama sphaerocarpa, were collected to study species differentiation using flow cytometry for 2C DNA assessment and molecular cytogenetics for karyotype organisation. All were 2n=48. Genome size ranged from 1.76 to 1.97 pg and revealed significant intraspecific variation correlated to the geographic distribution of the populations. The number and position of the two ribosomal gene families 5S and 45S were determined by fluorescent in situ hybridization, revealing chromosome reorganisation between species. In R. raetam and R. monosperma, the minor 5S loci co-localised with 45S on the satellite chromosome pair. Fluorochrome banding identified GC- and AT-rich DNA regions. In R. monosperma a unique chromomycin positive GC-rich band was observed associated with the secondary constriction. In contrast, an original pattern showing two chromomycin positive bands localised at each side of the extended rDNA locus was observed in R. sphaerocarpa and R. raetam. The polymorphism revealed in our cytogenetic data allowed us to separate the group of R. raetam and R. monosperma from R. sphaerocarpa