Background: Although the depth of the counting chamber is an important factor influencing sperm counting, no research has yet been reported on the measurement and comparison of the depth of the chamber. We measured the exact depths of six kinds of sperm counting chambers and evaluated their accuracy. Materials and Methods: In this prospective study, the depths of six kinds of sperm counting chambers for both manual and computer-aided semen analyses, including Makler (n=24), Macro (n=32), Geoffrey (n=34), GoldCyto (n=20), Leja (n=20) and Cell-VU (n=20), were measured with the Filmetrics F20 Spectral Reflectance Thin-Film Measurement System, then the mean depth, the range and the coefficient of variation (CV) of each chamber, and the mean depth, relative deviation and acceptability of each kind of chamber were calculated by the closeness to the nominal value. Among the 24 Makler chambers, 5 were new and 19 were used, and the other five kinds were all new chambers. Results: The depths (mean ± SD, μm) of Makler (new), Macro and Geoffrey chambers were 11.07 ± 0.41, 10.19 ± 0.48 and 10.00 ± 0.28, respectively, while those of GoldCyto, Leja and Cell-VU chambers were 23.76 ± 2.15, 20.49 ± 0.22 and 24.22 ± 2.58, respectively. The acceptability of Geoffrey chambers was the highest (94.12%), followed by Macro (65.63%), Leja (35%) and Makler (20%), while that of the other two kinds and the used Makler chamber was zero. Conclusion: There existed some difference between the actual depth and the corresponding nominal value for sperm counting chambers, and the overall acceptability was very low. Moreover, the abrasion caused by the long use, as of Makler chamber, for example, may result in unacceptability of the chamber. In order to ensure the accuracy and repeatability of sperm concentration results, the depth of the sperm counting chamber must be checked regularly.