Abstract The nucleotide diversity across 1705 bp of the G6pd gene is studied in 50 Drosophila melanogaster and 12 D. simulans lines. Our earlier report contrasted intraspecific polymorphism and interspecific differences at silent and replacement sites in these species. This report expands the number of European and African lines and examines the pattern of polymorphism with respect to the common A/B allozymes. In D. melanogaster the silent nucleotide diversity varies 2.8-fold across localities. The B allele sequences are two- to fourfold more variable than the derived A allele, and differences between allozymes are twice as among B alleles. There is strong linkage disequilibrium across the G6pd region. In both species the level of silent polymorphism increases from the 5′ to 3′ ends, while there is no comparable pattern in level of silent site divergence or fixation. The neutral model is not rejected in either species. Using D. yakuba as an outgroup, the D. melanogaster lineage shows a twofold greater rate of silent fixation, but less than half the rate of amino acid replacement. Lineage-specific differences in mutation fixation are inconsistent with neutral expectations and suggest the interaction of species-specific population size differences with both weakly advantageous and deleterious selection.