Prostate cancer is the most common cancer among men and the methods used to detect and diagnose prostate cancer are not sufficiently accurate. Radical prostatectomy is a surgical treatment of prostate cancer where the whole prostate is removed from the patient. Prostate tissue stiffness can be measured with a stiffness sensitive resonance sensor. The aim of this study was to measure the stiffness on the anterior and posterior side of fresh human prostate tissue in vitro and compare these two groups with each other and relate the findings with the prostate tissue histology. In a prostate tissue slice with mostly normal healthy tissue, the anterior side was significantly harder (p-value < 0.05) as expected. In a prostate tissue slice with areas of cancer tumors, no difference was found between the anterior and posterior sides. However, large stiffness variations were found within groups with measurements points on cancer tissue (coefficient of variation, CV = 42 and 85%), as opposed to groups without cancer tissue (CV = 27 and 28%). The large stiffness variations could be used as a sign for the presence of cancer. The results are promising for the development of an instrument and method for faster diagnosis on radical prostatectomy samples.