Coxsackievirus B3 (CVB3) has been identified as a major causative agent of acute and chronic myocarditis, but the involvement of other CVB serotypes in myocarditis has not been investigated. To dissect the pathological properties of different CVB serotypes toward primary cardiomyocytes, we tested their effects on primary cardiomyocyte cultures from neonatal rats. Morphological abnormalities were examined by both light and fluorescence microscopy after Hoechst 33342 staining, and loss of cell viability was estimated by MTT assay. All six CVB serotypes showed a similar degree of severe toxicity toward primary cardiomyocytes. CVB clinical isolates had cytopathic effects (CPEs) similar to those of their respective CVB reference strains. Within 1-2 days of infection with multiplicities of infection MOI 50, the cells began to experience morphological changes including cell shrinkage, rounding-up, and slight nuclear condensation. The irreversible loss of cell viability was readily observed within 3-5 days following virus infection. These results suggest that all six CVB serotypes induce direct, irreversible toxicity towards cardiomyocytes, which eventually leads to the death of infected cells. These findings indicate that the variations in CVB serotype are not the limiting factor determining the susceptibility of cardiomyocytes to CVB infection.