Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3–4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of equivalent cells.Author SummaryFor a gene to function properly, it must be active in the right place, at the right time, and in the right amount. Changes in any of these features can lead to observable differences between individuals and species and in some cases can lead to disease. We do not currently understand how the position, timing, and amount of gene expression is encoded in DNA sequence. One approach to this problem is to compare how gene expression differs between species and to try to relate changes in DNA sequence to changes in gene expression. Here, we take the first step by comparing gene expression patterns at high spatial and temporal resolution between embryos of three species of fruit flies. We develop methods for comparing gene expression in individual cells, which allow us to control for variation in the size, shape, and number of nuclei between embryos. We find measurable quantitative differences in the patterns for all individual genes that we have examined. However, by considering all genes in our dataset at once, we show that many genes are changing together, leading to largely equivalent types of cells in these three species.