A study of the variation in pattern and frequency of constitutive heterochromatin and nucleolar organizing regions of the X chromosomes of male Chorthippus parallelus grasshoppers in 25 populations within the Iberian peninsula requires us to revise our interpretation of the biogeography and evolutionary history of this species. Hybridization between the subspecies Cp erythropus and Cp parallelus, previously only known from populations in the Pyrenean cols, is shown to extend at least 400 km further into north-west Spain. A novel X-chromosome variant is described that appears to be close to fixation in 18 populations, mainly from the centre and south of Spain. Our findings indicate a possible independent origin for each of three distinct, nonderivative X variants present in Spain: the northern Cp erythropus and Cp parallelus variants, and a central-southern Cp erythropus variant. The first two are distinguished by interstitial and distal C bands, respectively, whereas the central-southern form has neither. This central-southern form is probably the current representative of the ancestral Iberian X variant. The pattern of variation supports the hypotheses of multiple refugia for Iberian populations and that more hybrid zones exist between these chromosomal variants.