Abstract Introduction Juvenile dermatomyositis (JDM) can be classified into clinical serological subgroups by distinct myositis-specific antibodies (MSAs). It is incompletely understood whether different MSAs are associated with distinct pathological characteristics, clinical disease activities, or response to treatment. Methods We retrospectively reviewed clinicopathological data from consecutive JDM patients followed in the pediatric rheumatology clinic at a single center between October 2016 and November 2018. Demographics, clinical data, and laboratory data were collected and analyzed. Detailed muscle biopsy evaluation of four domains (inflammation, myofiber, vessels, and connective tissue) was performed, followed by statistical analysis. Results Of 43 subjects included in the study, 26 (60.5%) had a detectable MSA. The most common MSAs were anti-NXP-2 (13, 30.2%), anti-Mi-2 (7, 16.3%), and anti-MDA-5 (5, 11.6%). High titer anti-Mi-2 positively correlated with serum CK > 10,000 at initial visit (r = 0.96, p = 0.002). Muscle biopsied from subjects with high titer anti-Mi-2 had prominent perifascicular myofiber necrosis and perimysial connective tissue damage that resembled perifascicular necrotizing myopathy, but very little capillary C5b-9 deposition. Conversely, there was no positive correlation between the levels of the anti-NXP-2 titer and serum CK (r = − 0.21, p = 0.49). Muscle biopsies from patients with anti-NXP-2 showed prominent capillary C5b-9 deposition; but limited myofiber necrosis. Only one patient had anti-TIF1γ autoantibody, whose muscle pathology was similar as those with anti-NXP2. All patients with anti-MDA-5 had normal CK and near normal muscle histology. Conclusions Muscle biopsy from JDM patients had MSA specific tissue injury patterns. These findings may help improve muscle biopsy diagnosis accuracy and inform personalized treatment of JDM.