I present here evidence of remarkable local changes in GC/AT substitution biases and in crossover frequencies on Drosophila chromosomes. The substitution pattern at 10 loci in the telomeric region of the X chromosome was studied for four species of the Drosophila melanogaster species subgroup. Drosophila orena and Drosophila erecta are clearly the most closely related species pair (the erecta complex) among the four species studied; however, the overall data at the 10 loci revealed a clear dichotomy in the silent substitution patterns between the AT-biased- substitution melanogaster and erecta lineages and the GC-biased-substitution yakuba and orena lineages, suggesting two or more independent changes in GC/AT substitution biases. More importantly, the results indicated a between- loci heterogeneity in GC/AT substitution bias in this small region independently in the yakuba and orena lineages. Indeed, silent substitutions in the orena lineage were significantly biased toward G and C at the consecutive yellow, lethal of scute, and asense loci, but they were significantly biased toward A and T at sta. The substitution bias toward G and C was centered in different areas in yakuba (significantly biased at EG:165H7.3, EG:171D11.2, and suppressor of sable). The similar silent substitution patterns in coding and noncoding regions, furthermore, suggested mutational biases as a cause of the substitution biases. On the other hand, previous study reveals that Drosophila yakuba has about 20-fold higher crossover frequencies in the telomeric region of the X chromosome than does D. melanogaster; this study revealed that the total genetic map length of the yakuba X chromosome was only about 1.5 times as large as that of melanogaster and that the map length of the X-telomeric y-sta region did not differ between Drosophila yakuba and D. erecta. Taken together, the data strongly suggested that an approximately 20- fold reduction in the X-telomeric crossover frequencies occurred in the ancestral population of D. melanogaster after the melanogaster-yakuba divergence but before the melanogaster-simulans divergence.