Scanning Acoustic Microscopy (SAM) is a powerful complement to the variety of available techniques for the investigation of auditory ossicles. The contrast of acoustic microscopy depends on the elastic response of material to acoustic waves and provides information about local changes in the acousto-mechanical properties, e.g. the acoustic impedance, at a microscopic scale. For SAM it is important to have a flat and very smooth surface. Therefore all auditory ossicles from 25 postmortem middle ears were embedded in PMMA and the sample surface was grinded and polished using successively finer diamond paste. Measurements were done in reflection mode with the scanning acoustic microscopes KSI SAM100 and KSI SAM2000, Kraemer Scientific Instruments, Herborn, Germany, at frequencies of 25 and 900 MHz, respectively. The data were analyzed by a custom made software, which yields calibrated impedance images of ossicle slices. At 25 MHz the mean acoustic impedances were 4.7 Mrayl for stapes, 6.7 Mrayl for incus and 7.1 Mrayl for malleus. By use of the Multi Layer Analysis technique the distribution of acoustic impedance can be calculated for the high frequency data at 900 MHz, also. Here the acoustic impedance values were 1.5 to 2 times lower compared to those at 25 MHz. Statistically significant differences of the acoustic impedance were found within the different ossicles (stapes, incus and malleus) and between different regions within individual ossicles (multi-variation analysis, p < 0.05).