Background: RNA-seq based on short reads generated by next generation sequencing technologies has become the main approach to study differential gene expression. Until now the main applications of this technique have been to study the variation of gene expression in a whole organism, tissue or cell type under different conditions or at different developmental stages. However, RNA-seq also has a great potential to be used in evolutionary studies to investigate gene expression divergence in closely related species. Since the more reliable statistical methods for differential gene expression inference are based on the use of raw read count data, the reference genomes of the species to be compared need to be highly comparable. Results: We show that the published genomes and annotations of the three closely related Drosophila species, D. melanogaster, D. simulans and D. mauritiana, have limitations for inter-specific gene expression studies. This is due to missing gene models in at least one of the genome annotations, unclear orthology assignments and significant length differences in the different species. We propose that published reference genomes should be re-annotated before using them as references for RNA-seq experiments to include as many genes as possible and to account for a potential length bias. For that we present a straight-forward reciprocal re-annotation pipeline that allows to reliably compare the expression for nearly all genes annotated in D. melanogaster. We carried out a RNA-seq experiment in combination with quantitative real-time PCR to confirm that the newly generated gene sets do not result in a high number of false positives as observed with references that still show a clear length difference of gene models between species. Conclusions: We conclude that our reciprocal re-annotation of previously published genomes facilitates the analysis of significantly more genes in an inter-specific differential gene expression study. We propose that the established pipeline can easily be applied to re-annotate other genomes of closely related animals and plants to improve comparative expression analyses. Overall design: mRNA profiles of larval eye-antennal imaginal discs (late L3) of three species of Drosophila (D. melanogaster OreR, D. simulans YVF and D. mauritiana TAM16) were generated by deep sequencing using Illumina HiSeq 2000. 3 biological replicates were generated for D. melanogaster sample and sequenced in 50 bp single-end reads; 3 biological replicates were generated for D. simulans sample and sequenced in 100 bp paired-end reads; 6 biological replicates were generated for D. mauritiana sample, 3 of these were sequenced in 50 bp single-end reads and the other 3 in 100 bp paired-end reads.