Enteroviruses like coxsackievirus B3 (CVB3) are common suspects in myocarditis/dilated cardiomyopathy patients. Autoimmunity has been proposed as an underlying mechanism, but direct evidence of its role is lacking. To delineate autoimmune response in CVB3 myocarditis, we used IA(k) dextramers for cardiac myosin heavy chain (Myhc)- 334-352. We have demonstrated that myocarditis-susceptible A/J mice infected with CVB3 generate Myhc–reactive CD4 T cells and such a repertoire was absent in nave mice as measured by proliferative response to Myhc- 334-352 and IA(k) dextramer staining. We also detected Myhc- 334-352 dextramer(+) cells in the hearts of CVB3-infected mice. The autoreactive T cell repertoire derived from infected mice contained a high frequency of interleukin-17-producing cells capable of inducing myocarditis in nave recipients. The data suggest that CVB3, a bona fide pathogen of cardiovascular system that primarily infects the heart can lead to the secondary generation of autoreactive T cells and contribute to cardiac pathology.