Background: Overlaps in spatial patterns of gene expression are frequently an initial clue to genetic interactions during embryonic development. However, manual inspection of images requires considerable time and resources impeding the discovery of important interactions because tens of thousands of images exist. The FlyExpress discovery platform was developed to facilitate data-driven comparative analysis of expression pattern images from Drosophila embryos. Results: An image-based search of the BDGP and Fly-FISH datasets conducted in FlyExpress yields fewer but more precise results than text-based searching when the specific goal is to find genes with overlapping expression patterns. We also provide an example of a FlyExpress contribution to scientific discovery: an analysis of gene expression patterns for multigene family members revealed that spatial divergence is far more frequent than temporal divergence, especially after the maternal to zygotic transition. This discovery provides a new clue to molecular mechanisms whereby duplicated genes acquire novel functions. Conclusions: The application of FlyExpress to understanding the process by which new genes acquire novel functions is just one of a myriad of ways in which it can contribute to our understanding of developmental and evolutionary biology. This resource has many other potential applications, limited only by the investigator's imagination. Developmental Dynamics 241:150–160, 2012. © 2011 Wiley Periodicals, Inc.