In autopsy practice, the thickness of ventricular walls is one of the parameters used to identify cardiac hypertrophy. The presented study aimed to compare ante- and postmortem measurements of ventricular wall thickness, (i) to determine a postmortem standardized localization and dissection method for ventricular wall measurements, and (ii) to determine the ability of postmortem measurements in recognition of antemortem hypertrophy. A single-center prospective study was conducted at the Institute of Legal Medicine in Hamburg, Germany. Sixty hearts were dissected alternating by the inflow-outflow or short-axis method, and the ventricular walls were measured at different locations and compared with the echocardiographic values of the end-diastolic phase during life of these individuals. The results showed measurement differences between the autoptic and echocardiographic values—for the left ventricle between 3.3 and 5.2 mm, for the right ventricle between 0.2 and 1.1 mm, and for the septum between 1.3 and 1.4 mm. Diagnostic performance of recognizing antemortem hypertrophy with postmortem measurement was poor, except for measuring the right ventricle and septum with the short-axis method (area under the ROC curve of 0.72 and 0.82, respectively). According to the results, cardiac changes may occur postmortem and need to be considered when used for diagnosing cardiac pathology. The postmortem diagnosis of left or right ventricular hypertrophy should always be made in conjunction with other, particularly cardiac, autopsy findings. An autoptic diagnosis of hypertrophy solely by a ventricular wall thickness > 15 mm or > 5 mm alone is not sufficient.