The in vivo mechanisms in chronic myocarditis remain unclear. The aim of the current study was to clarify the genomic difference of amyocarditic (CB3O) and myocarditic (CB3M) coxsackievirus B3 (CB3) and the pathogenesis of in vivo mechanisms in chronic myocarditis. We examined the histopathology of CB3-inoculated wild-type (WT) and severe combined immunodeficient (SCID) mice with and without adoptive transfer of lymphocytes. There were no differences in viral growth between CB3O and CB3M. There were four to six nucleotide differences in the sequence of CB3O in comparison with the known CB3M. The difference in virus sequence between CB3O and CB3M was very minimal. The changes were located in 1A, 1C, and 1D regions, which encode the structural capsid proteins. Definite myocarditis developed in WT C3H (H-2(k)) inoculated with CB3M. On the contrary, trivial or mild myocarditis occurred in WT C3H mice inoculated with CB3O. In SCID C3H and SCID C57BL/6 (H-2(b)) mice, definite myocarditis developed by inoculation with both CB3O and CB3M. Myocardial lesion was less severe in the mice infected with CB3O than in those with CB3M. After anti-CD8 antibody treatment, myocarditis was easily induced in mice originally showing resistance to infection. In addition, chronic myocarditis developed in CB3O-infected SCID C3H mice reconstituted with CB3M-sensitized splenocytes of WT C3H mice. The development of chronic myocarditis primarily depends on the presence or absence of the virus genome, and secondarily on the complex interaction between virus virulence and immunological background of the host. CB3 infection may cause chronic myocarditis with ongoing inflammation with or without viral persistence.