Enteroviruses (EVs) are amongst the most common human pathogens and are characterized by high genetic and phenotypic diversity. EV-D68, a respiratory EV, and EV-D94, an enteric EV, belong to the same species while displaying distinct phenotypic characteristics. Our results highlight temperature as a critical factor in limiting EV-D68 tropism and suggest that each virus have different strategies to deal with the host response. In parallel, we focus on dissecting the viral determinants behind EVs diversity by generating chimeric constructs between EV-D68 and EV-D94. Using a linear mathematical model, we identified the critical role of the origin of 5’UTR, 2A and P3 proteins as viability factors while characterization of the phenotype of viable chimeras points to the 2A protease as a major determinant of viral fitness and tropism. We hope that, by investigate EVs pathogenic diversity, this work can bring us closer to develop strategies to prevent or treat these infections.