Parasitic supernumerary (B) chromosomes show high capability to spread across populations. But the existence of abrupt discontinuities in their distribution demands an explanation. The grasshopper Eyprepocnemis plorans plorans harbour supernumerary chromosomes in all natural populations hitherto analyzed from the Circum-Mediterranean region, with the single exception of the headwaters of the Iberian Segura River and several of its tributaries. To ascertain the causes of this distribution pattern, we analyze here the genetic structure of five natural populations collected in this zone (two +B and three -B), by means of ISSR markers. We found significant population structure, with two kinds of populations coinciding with +B and -B ones, separated by strong barriers to gene flow. This gives strong support to the hypothesis that the non-B populations precede B origin, and that B-carrying individuals from coastal zones have been able to colonize upstream areas, until geographical barriers (usually narrow canyons and arid areas surrounding them) impeded their advance.