Heat shock protein 90 (Hsp90) is a protein chaperone that is upregulated and released from pancreatic β cells under pro-inflammatory conditions. We hypothesized that serum Hsp90 may have utility as a biomarker of type 1 diabetes risk and exhibit elevations before the onset of clinically significant hyperglycemia. To this end, total levels of the alpha cytoplasmic isoform of Hsp90 were assayed in autoantibody-positive progressors to type 1 diabetes using banked serum samples from the TrialNet Pathway to Prevention Cohort that had been collected 12 months prior to diabetes onset, with comparison to age, sex, and BMI-category matched autoantibody-positive nonprogressors and healthy controls. Hsp90 levels were higher in autoantibody-positive progressors and nonprogressors ≤ 18 years of age compared to matched healthy controls. However, Hsp90 levels were not different between progressors and nonprogressors in any age group. Hsp90 was positively correlated with age in control subjects, but this correlation was absent in autoantibody positive individuals. In aggregate these data indicate that elevated Hsp90 levels are present in youth with β cell autoimmunity, but are not able to distinguish youth or adult type 1 diabetes progressors from nonprogressors in samples collected 12 months prior to diabetes development.