A cytogenetic study of 62 populations of Santolina pectinata in Spain shows the existence of two ploidy levels. The diploid cytotypes with 2n = 18 occupy the eastern Betic mountains, and the tetraploid cytotypes with 2n = 36 are located on the spurs of the Iberian System. The former show a much wider ecological spectrum than the latter. Mixed cytotypes were observed in two diploid populations, with one tetraploid in each, showing different karyotypes. Three trisomic individuals were detected, one in a diploid population and the other two in a tetraploid population. Also, three hypotetraploid individuals were detected in a tetraploid population. Polyploidy is shown to be spontaneous and recurrent, promoting partial sterility in the pollen. Structural chromosomal changes, principally translocations, and local speciation through autopolyploidy are the principal factors in the evolution and diversification of this species. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 156, 657–667.