Hybrids have reduced fitness because of faulty interactions among genes that have diverged between the pure species. The most extreme hybrid defect is inviability. Hybrids therefore provide an easy to screen phenotype to identify what genes and molecular pathways have functionally diverged as genomes differentiate. Here I identify the Drosophila melanogaster allele of the highly conserved and essential gap gene giant (gt) as a key genetic determinant of hybrid inviability in crosses with D. santomea. The presence of this allele in D. melanogaster/D. santomea hybrids causes an abdominal ablation not seen in either pure species. giant is a key developmental regulator whose role in anterior-posterior specification of the dipteran embryo is conserved over 100 million years. The interaction of giant with a second allele, tailless,tll, is involved in the hybrid defects observed in these Drosophila hybrids. These results indicate that even genes with crucial developmental roles that are conserved over vast evolutionary time scales can experience functional evolution that leads to inviability of hybrids