Animal promoters initiate transcription either at precise positions (narrow 
promoters) or dispersed regions (broad promoters), a distinction referred to as 
promoter shape. Although highly conserved, the functional properties of 
promoters with different shapes and the genetic basis of their evolution remain 
unclear. Here we used natural genetic variation across a panel of 81 Drosophila 
lines to measure changes in transcriptional start site (TSS) usage, identifying 
thousands of genetic variants affecting transcript levels (strength) or the 
distribution of TSSs within a promoter (shape). Our results identify promoter 
shape as a molecular trait that can evolve independently of promoter strength. 
Broad promoters typically harbor shape-associated variants, with signatures of 
adaptive selection. Single-cell measurements demonstrate that variants 
modulating promoter shape often increase expression noise, whereas heteroallelic 
interactions with other promoter variants alleviate these effects. These results 
uncover new functional properties of natural promoters and suggest the 
minimization of expression noise as an important factor in promoter evolution.