This is the final version of the article. Available from the publisher via the DOI in this record. ; We present SuperFly (http://superfly.crg.eu), a relational database for quantified spatio-temporal expression data of segmentation genes during early development in different species of dipteran insects (flies, midges and mosquitoes). SuperFly has a special focus on emerging non-drosophilid model systems. The database currently includes data of high spatio-temporal resolution for three species: the vinegar fly Drosophila melanogaster, the scuttle fly Megaselia abdita and the moth midge Clogmia albipunctata. At this point, SuperFly covers up to 9 genes and 16 time points per species, with a total of 1823 individual embryos. It provides an intuitive web interface, enabling the user to query and access original embryo images, quantified expression profiles, extracted positions of expression boundaries and integrated datasets, plus metadata and intermediate processing steps. SuperFly is a valuable new resource for the quantitative comparative study of gene expression patterns across dipteran species. Moreover, it provides an interesting test set for systems biologists interested in fitting mathematical gene network models to data. Both of these aspects are essential ingredients for progress toward a more quantitative and mechanistic understanding of developmental evolution. ; MEC-EMBL agreement for the EMBL/CRG Research Unit in Systems Biology [to research group of Johannes Jaeger]; BioPreDyn Consortium (European Commission Grant FP7-KBBE-2011-5/289434); Catalan funding agency AGAUR [SGR Grant 406]; Spanish Ministerio de Economia y Competitividad (MINECO, formerly MICINN) [BFU2009-10184, BFU2012-33775]; MINECOfunded infrastructure technician support [ref.: PTA2010- 4446-I to bioinformatics core and Toni Hermoso-Pulido in particular]. MINECO, Centro de Excelencia Severo Ochoa [2013-2017, SEV-2012-0208 to The Centre for Genomic Regulation (CRG)]. Funding for open access charge: MECEMBL agreement for the EMBL/CRG Research Unit in Systems Biology