DNA bending is critical for DNA packaging, recognition, and repair, and occurs toward either the major or the minor groove. The anisotropy of B-DNA groove bending was quantified for eight DNA sequences by free energy simulations employing a novel reaction coordinate. The simulations show that bending toward the major groove is preferred for non-A-tracts while the A-tract has a high tendency of bending toward the minor groove. Persistence lengths were generally larger for bending toward the minor groove, which is thought to originate from differences in groove hydration. While this difference in stiffness is one of the factors determining the overall preference of bending direction, the dominant contribution is shown to be a free energy offset between major and minor groove bending. The data suggests that, for the A-tract, this offset is largely determined by inherent structural properties, while differences in groove hydration play a large role for non-A-tracts. By quantifying the energetics of DNA groove bending and rationalizing the origins of the anisotropy, the calculations provide important new insights into a key biological process.