Many disease‐relevant phenotypes modeled in inbred mice have been shown to be strain‐dependent, indicating the important influence of genetic background on disease phenotypes. Although C57BL/6 mice are one of the most commonly used inbred strains in laboratory research, there are multiple substrains (eg, B6J vs B6N) that have been separated for more than 50 years. Thus, understanding the substrain differences is important for scientific rigor and reproducibility. In this study, seizure susceptibility, spontaneous seizures, and survival were compared between Scn1a^+/− mice on (C57BL/6J × 129S6/SvEvTac)F1 (F1J) vs (C57BL/6N × 129S6/SvEvTac)F1 (F1N) strain backgrounds. F1N.Scn1a^+/− mice were more susceptible to hyperthermia‐induced seizures, yet had milder spontaneous seizures and improved survival relative to F1J.Scn1a^+/− mice. Our results indicate that choice of C57BL/6 substrain may significantly alter disease phenotypes and should be considered carefully in experimental design using the Scn1a^+/− Dravet mouse model, as well as other mouse models of epilepsy.