Background: Prospero (Hyacinthaceae) provides a unique system to assess the impact of genome rearrangements on plant diversification and evolution. The genus exhibits remarkable chromosomal variation but very little morphological differentiation. Basic numbers of x = 4, 5, 6 and 7, extensive polyploidy, and numerous polymorphic chromosome variants were described, but only three species are commonly recognized: P. obtusifolium, P. hanburyi, and P. autumnale s.l., the latter comprising four diploid cytotypes. The relationship between evolutionary patterns and chromosomal variation in diploids, the basic modules of the extensive cytological diversity, is presented. Results: Evolutionary inferences were derived from fluorescence in situ hybridization (FISH) with 5S and 35S rDNA, genome size estimations, and phylogenetic analyses of internal transcribed spacer (ITS) of 35S rDNA of 49 diploids in the three species and all cytotypes of P. autumnale s.l. All species and cytotypes possess a single 35S rDNA locus, interstitial except in P. hanburyi where it is sub-terminal, and one or two 5S rDNA loci (occasionally a third in P. obtusifolium) at fixed locations. The localization of the two rDNA types is unique for each species and cytotype. Phylogenetic data in the P. autumnale complex enable tracing of the evolution of rDNA loci, genome size, and direction of chromosomal fusions: mixed descending dysploidy of x = 7 to x = 6 and independently to x = 5, rather than successive descending dysploidy, is proposed. Conclusions: All diploid cytotypes are recovered as well-defined evolutionary lineages. The cytogenetic and phylogenetic approaches have provided excellent phylogenetic markers to infer the direction of chromosomal change in Prospero. Evolution in Prospero, especially in the P. autumnale complex, has been driven by differentiation of an ancestral karyotype largely unaccompanied by morphological change. These new results provide a framework for detailed analyses of various types of chromosomal rearrangements and karyotypic variation in polyploids.