Identifying the amino acid changes responsible for electrophoretic variants is essential to understanding the significance of allozyme polymorphism in adaptation. The amino acid mutations responsible for the common G6PD allozyme polymorphisms in Drosophila melanogaster have been recently described. This study characterizes the amino acid changes associated with 11 rare electrophoretic G6PD variants. The 11 rare electrophoretic variants result from six independent amino acid mutations. The in vivo function of the rare variants was determined in an earlier study and most variants fell into one of two function classes. It is shown here that the function of the rare variants reflects the state of the Pro/Leu mutation responsible for the A/B allozyme polymorphism in each variant. Two mutations destabilize quaternary structure resulting in shifts from tetrameric dimeric alleles, and one of these also results in a variant with in vivo function intermediate to A and B. That mutation is an aspartic-acid-to-asparagine change that is two residues away from the Pro/Leu polymorphism responsible for the A/B dimertetramer quaternary shift. Structure-function relationships based on studies of human G6PD deficiency-associated mutations predict that these last two amino acid changes fall within the protein domain responsible for NADP binding.