The evolution of canalized traits is a central question in evolutionary biology. Natural variation in highly conserved traits can provide clues about their evolutionary potential. Here we investigate natural variation in a conserved trait—even-skipped (eve) expression at the cellular blastoderm stage of embryonic development in Drosophila melanogaster. Expression of the pair-rule gene eve was quantitatively measured in three inbred lines derived from a natural population of D. melanogaster. One line showed marked differences in the spacing, amplitude and timing of formation of the characteristic seven-striped pattern over a 50-min period prior to the onset of gastrulation. Stripe 5 amplitude and the width of the interstripe between stripes 4 and 5 were both reduced in this line, while the interstripe distance between stripes 3 and 4 was increased. Engrailed expression in stage 10 embryos revealed a statistically significant increase in the length of parasegment 6 and a decrease in the length of parasegments 8 and 9. These changes are larger than those previously reported between D. melanogaster and D. pseudoobscura, two species that are thought to have diverged from a common ancestor over 25 million years ago. This line harbors a rare 448 bp deletion in the first intron of knirps (kni). This finding suggested that reduced Kni levels caused the deviant eve expression, and indeed we observed lower levels of Kni protein at early cycle 14A in L2 compared to the other two lines. A second of the three lines displayed an approximately 20% greater level of expression for all seven eve stripes. The three lines are each viable and fertile, and none display a segmentation defect as adults, suggesting that early-acting variation in eve expression is ameliorated by developmental buffering mechanisms acting later in development. Canalization of the segmentation pathway may reduce the fitness consequences of genetic variation, thus allowing the persistence of mutations with unexpectedly strong gene expression phenotypes.