Alloreactivity is the response of T cells to MHC molecules not encountered during thymic development. A small population (1-8%) of peripheral T cells in mice and humans express two TCRs due to incomplete allelic exclusion of TCRα, and we hypothesized they are highly alloreactive. FACS analysis of mouse T cell MLR revealed increased dual TCR T cells among alloreactive cells. Quantitative assessment of the alloreactive repertoire demonstrated a nearly 50% reduction in alloreactive T cell frequency among T cells incapable of expressing a secondary TCR. We directly demonstrated expansion of the alloreactive T cell repertoire at the single cell level by identifying a dual TCR T cell with distinct alloreactivities for each TCR. The importance of dual TCR T cells is clearly demonstrated in a parent-into-F1 model of graft-vs-host disease, where dual TCR T cells comprised up to 60% of peripheral activated T cells, demonstrating a disproportionate contribution to disease.