Clinal variation has been described in a number of inversions in Drosophila but these clines are often characterized by cytological techniques using small sample sizes, and associations with specific genes are rarely considered. Here we have developed a molecular assay for In(3R)Payne in Drosophila melanogaster from eastern Australia populations. It shows in repeated samples that the inversion cline is very tightly associated with latitude and is almost fixed in tropical populations while relatively rare in temperate populations. This steep cline has shifted in position in the last 20 years. The heat shock gene, hsr-omega, located centrally inside the inversion sequence, shows a different clinal pattern to In(3R)Payne. These results suggest strong ongoing selection on In(3R)Payne over the last 100 years since the colonization of Australia that is partly independent of hsr-omega.