Betanodaviruses are the causative agents of viral nervous necrosis and affect a broad range of fish species worldwide. Their bi-segmented genome is composed of the RNA1 and the RNA2 molecules encoding the viral polymerase and the coat protein, respectively. In southern Europe the presence of the RGNNV and the SJNNV genotypes, and the RGNNV/SJNNV and RGNNV/SJNNV reassortants has been documented. Several studies have reported a correlation between water temperature and disease onset. To explore the replication efficiency of betanodaviruses with different genomes in relation to temperature and to understand the role of genetic reassortment on viral phenotype, RGNNV, SJNNV, RGNNV/SJNNV and RGNNV/SJNNV field isolates were fully sequenced, and growth curves generated in vitro at four different temperatures (15, 20, 25, 30 °C) were developed for each isolate. The data obtained, corroborated by statistical analysis, demonstrated that viral titres of diverse betanodavirus genotypes varied significantly in relation to the incubation temperature of the culture. In particular, at 30 °C betanodaviruses under investigation presented different phenotypes, and viruses containing the RNA1 of the RGNNV genotype showed the best replication efficiency. Laboratory results demonstrated that viruses clustering within the same genotype based on the polymerase gene, possess similar growth kinetics in response to temperature, thus highlighting the key role of RNA1 in controlling viral replication at different environmental conditions. The results generated might have practical implications for the inference of viral phenotype according to genetic features and may contribute to a better understanding of betanodavirus ecology.