Restriction-site polymorphism at the rp49 gene region has been studied in 234 lines of Drosophila subobscura representing different gene arrangements for the O chromosome. The population surveyed (El Pedroso, Spain) was sampled four times in each of two consecutive years. The data indicate that the two chromosomal classes studied, O[ST] and O[3 + 4], are genetically differentiated in El Pedroso. Comparison of the present results with those previously obtained for two other populations further supports that, for a given chromosomal class, European populations are not genetically differentiated. This lack of differentiation at the rp49 region within O[ST] and within O[3 + 4] stands in contrast to the clear latitudinal clines found in Europe for these arrangements.