Objective: To define the clinicopathologic features and diagnostic utility associated with anti-cytosolic 5′-nucleotidase 1A (NT5C1A) antibody seropositivity in idiopathic inflammatory myopathies (IIMs). Methods: Anti-NT5C1A antibody status was clinically tested between 2014 and 2019 in the Washington University neuromuscular clinical laboratory. Using clinicopathologic information available for 593 patients, we classified them as inclusion body myositis (IBM), dermatomyositis, antisynthetase syndrome, immune-mediated necrotizing myopathy (IMNM), nonspecific myositis, or noninflammatory muscle diseases. Results: Of 593 patients, anti-NT5C1A antibody was found in 159/249 (64%) IBM, 11/53 (21%) dermatomyositis, 7/27 (26%) antisynthetase syndrome, 9/76 (12%) IMNM, 20/84 (24%) nonspecific myositis, and 6/104 (6%) noninflammatory muscle diseases patients. Among patients with IBM, anti-NT5C1A antibody seropositive patients had more cytochrome oxidase-negative fibers compared with anti-NT5C1A antibody seronegative patients. Among 14 IBM patients initially negative for anti-NT5C1A antibody, three patients (21%) converted to positive. Anti-NT5C1A antibody seropositivity did not correlate with malignancy, interstitial lung disease, response to treatments in dermatomyositis, antisynthetase syndrome, and IMNM, or survival in IIMs. Interpretation: Anti-NT5C1A antibody is associated with IBM. However, the seropositivity can also be seen in non-IBM IIMs and it does not correlate with any prognostic factors or survival.