Published online ; Journal Article ; Research Support, Non-U.S. Gov't ; This is the final version of the article. Available from BioMed Central via the DOI in this record. ; BACKGROUND: Modern sequencing technologies have massively increased the amount of data available for comparative genomics. Whole-transcriptome shotgun sequencing (RNA-seq) provides a powerful basis for comparative studies. In particular, this approach holds great promise for emerging model species in fields such as evolutionary developmental biology (evo-devo). RESULTS: We have sequenced early embryonic transcriptomes of two non-drosophilid dipteran species: the moth midge Clogmia albipunctata, and the scuttle fly Megaselia abdita. Our analysis includes a third, published, transcriptome for the hoverfly Episyrphus balteatus. These emerging models for comparative developmental studies close an important phylogenetic gap between Drosophila melanogaster and other insect model systems. In this paper, we provide a comparative analysis of early embryonic transcriptomes across species, and use our data for a phylogenomic re-evaluation of dipteran phylogenetic relationships. CONCLUSIONS: We show how comparative transcriptomics can be used to create useful resources for evo-devo, and to investigate phylogenetic relationships. Our results demonstrate that de novo assembly of short (Illumina) reads yields high-quality, high-coverage transcriptomic data sets. We use these data to investigate deep dipteran phylogenetic relationships. Our results, based on a concatenation of 160 orthologous genes, provide support for the traditional view of Clogmia being the sister group of Brachycera (Megaselia, Episyrphus, Drosophila), rather than that of Culicomorpha (which includes mosquitoes and blackflies). ; Toni Hermoso Pulido from the CRG Bioinformatics Core provided help and support with the diptex database. We thank Debayan Datta, Maik Zehnsdorf, and Anna Menoyo (CRG Genomics Unit) for technical help. We gratefully acknowledge Urs Schmidt-Ott, for providing fly cultures, for sharing Episyrphus balteatus transcriptome data, for crucial advice on sequencing strategy, fly husbandry, and other experimental protocols, as well as for useful comments on the manuscript. Victor Jiménez-Guri drew the embryo pictures in Figure 1. This research was funded by the MEC/EMBL agreement for the EMBL/CRG Research Unit in Systems Biology, by AGAUR SGR grant 406, and by Grants BFU2009-10184 and BFU2009-09168 from the Spanish Ministry of Science and Innovation (MICINN). EJG is supported by ERASysBio+ Grant P#161 (MODHEART). LC was supported by grant PTA2011-6729-I from the Spanish Ministry of Science and Innovation (MICINN). JHC is supported by a Juan de la Cierva postdoctoral fellowship from the Spanish Ministry of Science and Innovation (JCI2010-07614). HK was supported by GABI-FUTURE grant BeetSeq (0315069A) by the German Federal Ministry of Education and Research.