The diagnosis of Type 2 myotonic dystrophy (DM2/proximal myotonic myopathy) is often overlooked because of a nonspecific clinical presentation and muscle biopsy findings of a "denervation-like" pattern of unknown specificity that combines increased fiber size variation, central nucleation, small angulated fibers, Type 2 fiber atrophy, and nuclear clumps. We determined the presence of these features in 104 patients designated as having an unidentified myopathy from a series of 2,100 muscle biopsies. Because CCUG expansions form pathogenic ribonuclear accumulations that can be detected by in situ hybridization, we validated and then used automated (CCUG)8 in situ hybridization as a reference standard to evaluate the value of each histologic feature for DM2 detection, identifying 8 DM2-positive and 96 DM2-negative cases. Multivariate analyses identified the combination of Type 2 fiber atrophy and central nucleation as the most predictive of DM2 (sensitivity, 1.0; specificity, 0.92). These features were mutually exclusive in non-DM2 patients (p < 0.0001). The relevance of this combination of features was confirmed in an additional independent series (15 DM2-positive vs 17 DM2-negative). Further investigation revealed that central nucleation selectively affects Type 2 fibers in DM2 and, conversely, that it affects Type 1 fibers in DM1 (p < 0.0001). These results will facilitate the routine detection of DM2 and further support the concept that DM2 has a distinct pathophysiology involving type 2 myofibers.