The reasons for the different outcome of coxsackievirus B3 (CVB3)-induced heart disease in humans are not well understood. Since there are no experimental data on the course of disease after infection with genetically different CVB3 in a natural variable population until now, we studied the outcome of virus infection in outbred NMRI mice after inoculation of genetically different CVB3 variants. Adult male mice were inoculated with seven closely related CVB3 variants. The histopathological changes of heart and pancreas tissue, antibody induction, virus titers, and persistence of viral positive- as well as negative-strand RNA in spleen and heart tissue were compared at day 7 or day 28 after infection to detect prerequisites and predictive factors for chronic myocarditis. Six CVB3 variants infected NMRI mice. CVB3 infection (i) did not induce detectable myocardial injury, (ii) caused signs of healing up acute myocarditis or (iii) ongoing chronic myocarditis. Neither IgG antibody responses nor the extent of destruction of exocrine pancreatic tissue or viral RNA load in spleen did correlate with myocardial histopathology. In contrast, a high persistent viral RNA load in heart tissue specimens was characteristic for mice developing chronic myocarditis. The results of the present study corroborate high viral load in the acute stage of myocarditis and high amounts of persisting CVB3 RNA in heart tissue as predictive marker of chronic myocarditis. The outcome of CVB3-induced heart disease in outbred NMRI mice depends strongly on the viral genetic background. In particular an important role of viral capsid proteins is suggested.