Type 1 diabetes (T1D) results from T cell–mediated destruction of insulin-producing β-cells. Insulin represents a key self-antigen in disease pathogenesis, as recent studies identified proinsulin-responding T cells from inflamed pancreatic islets of organ donors with recent-onset T1D. These cells respond to an insulin B-chain (InsB) epitope presented by the HLA-DQ8 molecule associated with high T1D risk. Understanding insulin-specific T-cell frequency and phenotype in peripheral blood is now critical. We constructed fluorescent InsB_10–23:DQ8 tetramers, stained peripheral blood lymphocytes directly ex vivo, and show DQ8⁺ patients with T1D have increased tetramer⁺ CD4⁺ T cells compared with HLA-matched control subjects without diabetes. Patients with a shorter disease duration had higher frequencies of insulin-reactive CD4⁺ T cells, with most of these cells being antigen experienced. We also demonstrate that the number of insulin tetramer⁺ effector memory cells is directly correlated with insulin antibody titers, suggesting insulin-specific T- and B-cell interactions. Notably, one of four control subjects with tetramer⁺ cells was a first-degree relative who had insulin-specific cells with an effector memory phenotype, potentially representing an early marker of T-cell autoimmunity. Our results suggest that studying InsB_10–23:DQ8 reactive T-cell frequency and phenotype may provide a biomarker of disease activity in patients with T1D and those at risk.