Lack of knowledge about how regulatory regions evolve in relation to their structure-function may limit the utility of comparative sequence analysis in deciphering cis-regulatory sequences. To address this we applied reverse genetics to carry out the first functional genetic complementation analysis of a eukaryotic cis-regulatory module - the even-skipped stripe 2 enhancer - from four Drosophila species. The functional evolution of this enhancer is non-clocklike: important functional differences have evolved between closely related species that are not found between distantly related species. We can attribute the functional conservation between distantly related species to evolutionary convergence rather than evolutionary stasis. Functional divergence of the stripe2 enhancer between closely related species is attributable to differences in activation levels rather than spatio-temporal control of gene expression. Our findings have implications for understanding enhancer structure-function, mechanisms of speciation, and computational identification of regulatory modules.