Restriction map variation in 64 X chromosome lines extracted from three different populations of Drosophila melanogaster was investigated with seven six-nucleotide-recognizing restriction enzymes for a 106-kb region encompassing the yellow gene and the achaete-scute complex that is located in the region of reduced crossing over close to the telomere. Nine restriction site polymorphisms (out of 176 sites scored) and 19 length polymorphisms (15 insertions and 4 deletions) were detected. The estimated level of heterozygosity per nucleotide, H = 0.0003, is much lower than that reported for autosomal and sex-linked loci located in regions with normal levels of crossing over. The overall frequency of polymorphic restriction sites is reduced. Six out of nine restriction site polymorphisms are unique and the other three have frequencies less than 0.17. Some large insertions have reached relatively high frequencies, 0.08 to 0.17. Consistent with the theoretically predicted negative relationship between crossing over and the magnitude of linkage disequilibrium, an increase in the relative number of nonrandom associations was observed in the y-ac-sc region.