The Resedaceae, containing 6 genera and ca. 85 species, are widely distributed in the Old World, with a major center of species diversity in the Mediterranean basin. Phylogenetic analyses of ITS and plastid trnL-trnF sequences of 66 species from all genera of the Resedaceae reveal (1) monophyly of the family, in congruence with preliminary phylogenetic studies; (2) molecular support for the traditional morphological subdivision of the Resedaceae into three tribes according to ovary and placentation types, and carpel number; (3) two monophyletic genera (Caylusea, Sesamoides), and one natural group (core Reseda), which includes the remaining four genera of the family (Ochradenus, Oligomeris, Randonia, Reseda); (4) a monophyletic origin for four of the six taxonomic sections recognized within Reseda (Leucoreseda, Luteola, Glaucoreseda, Phyteuma). Our results lead us to interpret an increment of the basic chromosome number in the family from x=5 to x=6 in at least two independent instances, and a broad representation of polyploids in multiple lineages across phylogenies, including association between octoploids and alien invasion in many parts of the world. Species diversity, endemism number, phylogenetic relationships and sequence divergence in Resedaceae suggest two major centers of differentiation, one in the western Mediterranean, and the other in the eastern Mediterranean and SW Asia. Two independent colonization events to the Canary Islands from Africa are indicated for the two Canarian Reseda endemics.