The C57BL/6 mouse strain is one of the most commonly used in experimental research. It is known to differ from other strains in baseline cardiovascular phenotypes as well as in response to pressure overload induced by aortic constriction. Since the generation of the C57BL/6 mouse line over a century ago, multiple substrains have been generated from the original. To identify potential substrain specific differences in response to pressure overload, we evaluated the effects of transverse aortic constriction (TAC) on survival, cardiac function, and expression of hypertrophic markers in three commonly used C57BL/6 substrains: C57BL/6J (JL), C57BL/6NCrl (CL), and C57BL/6NTac (TF). Survival and cardiac function were significantly lower in the CL and TF substrains compared with JL mice after TAC. Furthermore, the heart weight and lung weight as well as the expression of the hypertrophic marker Bnp were significantly greater in the CL mice compared with the JL. Histological assessment revealed marked left ventricular dilatation of CL and TF hearts while JL hearts showed increased wall thickness without dilatation. Our data demonstrate that cardiac response to pressure overload is distinct among the three commonly used C57BL/6 substrains of mice, which raises a cautionary note in study design and data interpretation.