We showed earlier that a transition mutation U234C, located within the completely conserved 5 nucleotide (nt) tract 5′-CGUUA (nt232–236) in the 5′ non-translated region (NTR) of the coxsackievirus B3 (CVB3) genome, attenuated CVB3 cardiovirulence in mice. To further explore the role of the sequence, we induced two single and one double transversion mutations in the conserved 5mer in a cardiovirulent CVB3 genome. The mutated sites partially or totally reverted to parental wild-type when progeny viruses were passaged at 37 °C, but remained stable when transfection and subsequent passages were performed at 33.5 °C. Viral replication in cell culture was attenuated at 37 °C or 39.5 °C relative to replication at 33.5 °C. While Western blot analysis demonstrated the level of protein translation consistent with virus replication, the ratios of positive to negative strand viral RNA at 37 °C in murine cells demonstrated a 2–5 fold diminution from those measured at 33.5 °C. Mutant CVB3 strains failed to replicate productively when inoculated into mice. The biological data are consistent with an hypothesis that proposes a lesion with primary effects at the level of positive strand viral RNA synthesis that results in attenuation of viral replication at physiologic temperature.