Studies of life rely on classifying organisms into species. However, since Darwin, there is no agreement about how to separate species from varieties. Contrary to a frequent belief, quantitative standards for species delineation are lacking and overdue, and debates about species delimitation create obstacles for conservation biology, agriculture, legislation, and education. To tackle this key biological question, we have chosen butterflies as model organisms. We sequenced and analyzed transcriptomes of 188 specimens representing pairs of close but clearly distinct species, populations, and taxa that are debated among experts. We find that species are robustly separated from populations by the combination of two measures computed on Z-linked genes: fixation index that detects hiatus between species, and the extent of gene flow that quantifies reproductive isolation. These criteria suggest that all 9 butterfly pairs that caused experts' disagreement are distinct species, not populations. When applied to Homo, our criteria agree that all modern humans are the same species distinct from Neanderthals, suggesting relevance of this study beyond butterflies. Furthermore, we found that proteins involved in interactions with DNA (including proteins encoded by trans-regulatory elements), circadian clock, pheromone sensing, development, and immune response recurrently correlate with speciation. A significant fraction of these proteins is encoded by the Z-chromosome, which appears to be resistant to introgression. Taken together, we find common speciation mechanisms in butterflies, reveal the central role of Zchromosome in speciation, and suggest quantitative criteria for species delimitation using genomic data, which is vital for the exploration of biodiversity.