Hsp90 is regarded as one of the best candidates for an evolved mechanism that regulates the expression of genetic and phenotypic variability. We examined nucleotide diversity in both the promoter and coding regions of Hsp90, the gene which encodes Hsp90 in Drosophila, in natural populations of Drosophila melanogaster from eastern Australia. We found that Hsp90 is polymorphic for only two nonsynonymous changes in the coding region, both of which are deletions of a lysine residue. One of these lysine deletions was in complete linkage disequilibrium with the inversion In(3L)P, and showed a significant association with latitude. The other lysine deletion reported here for the first time varied from 0 to 15% in natural populations, but did not show a clinal pattern. The regulatory and coding regions of Hsp90 showed very low nucleotide diversity compared to other nuclear genes, and chromosomes containing In(3L)P had lower levels of nucleotide diversity than the standard arrangements. Non-neutral evolution of Hsp90 was not supported by analyses of either the regulatory or coding regions of the gene. These results are discussed within the context of Hsp90 variation being involved in thermotolerance as well as the expression of genetic and phenotypic variability.