Objective To investigate the range of normal values of tympanometry of guinea pigs and the possible differences associated with genders and ears. Methods Included in this study were 40 guinea pigs with nor-mal hearing and no ear diseases. Under the anesthetized condition, each guinea pig's tympanogram was recorded, and their values were studied associated with different genders and ears. Results The tympanograms thus obtained were all type A. For all the tympanograms of 80 ears, the equivalent canal volume was about 0.09±0. 02 ml, the static compliance was 0.10 ±0.03 ml, the gradient was 0.14 ±0.06 ml, and the pressure of the peak admittance was -99.44±75.06 daPa . The findings of different gender groups yielded statistic differences in the values of e-quivalent canal volume (P=0. 012) but not in static compliance, gradient and peak admittance pressure (P>0. 05). Typical tympanograms were recorded for the both ears in 40 guinea pigs at the same time. There were no binaural differences (P>0.05). Conclusion The results show significant differences in the equivalent canal volumes be-tween male and female guinea pigs. These tympanograms may serve as a guide to detect middle ear dysfunctions in guinea pigs.