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The small heat shock gene (shsp) cluster of Drosophila buzzatii was sequenced and the gene order and DNA sequence were compared with those of the shsps in Drosophila melanogaster. The D. buzzatii shsp cluster contains an inversion and a duplication of hsp26. A phylogenetic tree was constructed based on hsp26 genes from several Drosophila species of the Sophophora and Drosophila subgenera. The tree shows first a separation of the Sophophora and the Drosophila subgenera and then the Drosophila subgenus is divided into the Hawaiian Drosophila and the repleta/virilis groups. Only the latter contain a duplicated hsp26. Comparing the gene organisation of the shsp cluster shows that all the Drosophila subgenus species contain the inversion. Putative heat shock elements (HSE) were found in the promoters of all the shsp and putative regulator elements for tissue specific expression were found in the promoter of hsp23, hsp27 and one of the hsp26 genes. hsp23 was found to be polymorphic for four non-synonymous changes that all lead to exchange of a Valine. The duplicated hsp26 gene in D. buzzatii (phsp26) was polymorphic for two non-synonymous changes. The allele frequencies of these variants were determined in nine D. buzzatii populations covering most of its distribution in Australia using high-resolution melting curves. The allele frequencies of one of the hsp23 variants showed a significant linear regression with longitude and the pooled frequency of the four Valine changes of hsp23 in the nine populations showed a significant linear regression with longitude and with a composite measure of climatic variables.