Structural deformations of DNA play a central role in many biological processes, including gene expression. The structural deformations are sensitive to the material properties of the molecule, and these, in turn, vary along the molecule’s length according to its base-pair sequence. Examples of “sequence-dependent” material properties include the stress-free curvature and the stiffness for bending and torsion. Quantifying and separating these sequence-dependent properties from experimental data remains a significant challenge as they often work in unison in nature. In this paper, we offer a method for resolving and quantifying the sequence-dependent stiffness of DNA from cyclization (loop closure) experiments using a computational rod model of the molecule.