Background: Mutation rates vary across the genome. Many trans factors that influence mutation rates have been identified, as have specific sequence motifs at the 1–7-bp scale, but cis elements remain poorly characterized. The lack of understanding regarding why different sequences have different mutation rates hampers our ability to identify positive selection in evolution and to identify driver mutations in tumorigenesis. Results: Here, we use a combination of synthetic genes and sequences of thousands of isolated yeast colonies to show that intrinsic DNA curvature is a major cis determinant of mutation rate. Mutation rate negatively correlates with DNA curvature within genes, and a 10% decrease in curvature results in a 70% increase in mutation rate. Consistently, both yeast and humans accumulate mutations in regions with small curvature. We further show that this effect is due to differences in the intrinsic mutation rate, likely due to differences in mutagen sensitivity and not due to differences in the local activity of DNA repair. Conclusions: Our study establishes a framework for understanding the cis properties of DNA sequence in modulating the local mutation rate and identifies a novel causal source of non-uniform mutation rates across the genome.