Microrheology has emerged as a powerful approach for elucidating mechanical properties of soft materials and complex fluids, especially biomaterials. In this technique, embedded microspheres are used to determine viscoelastic properties via generalized Stokes−Einstein relations, which assume the material behaves as a homogeneous continuum on the length scale of the probe. However, this condition can be violated if macromolecular systems form characteristic length scales that are larger than the probe size. Here we report observations of the onset of this effect in DNA solutions. We use microspheres driven with optical tweezers to determine the frequency dependence of the linear elastic and viscous moduli and their dependence on probe radius and DNA length. For well-entangled DNA, we find that the threshold probe radius yielding continuum behavior is ∼3× the reptation tube diameter, consistent with recent theoretical predictions. Notably, this threshold is significantly larger than the mesh size of the polymer network, and larger than typical probe sizes used in microrheology studies.