Widespread pigmentation diversity coupled with a well-defined genetic system of melanin synthesis and patterning in Drosophila provides an excellent opportunity to study phenotypes undergoing evolutionary change. Pigmentation variation is highly correlated with different ecological variables and is thought to reflect adaptations to different environments. Several studies have linked candidate genes from D. melanogaster to intra-population variation and interspecific morphological divergence, but less clearly to variation among populations forming pigmentation clines. We characterised a new thoracic trident pigmentation cline in D. melanogaster populations from eastern Australia, and applied a candidate gene approach to explain the majority of the geographically structured phenotypic variation. More melanised populations from higher latitudes tended to express less ebony than their tropical counterparts, and an independent artificial selection experiment confirmed this association. By partitioning temperature dependent effects, we showed that the genetic differences underlying clinal patterns for trident variation at 25°C do not explain the patterns observed at 16°C. Changes in thoracic trident pigmentation could be a common evolutionary response to climatically-mediated environmental pressures. On the Australian east coast most of the changes appear to be associated with regulatory divergence of the ebony gene but this depends on temperature.