Semen of all bulls is not suitable for freezing; similarly, all ejaculates with excellent sperm vitality are not suitable for freezing. Based on sperm survival at post-thawing, bull ejaculates are categorized as good freezability ejaculates (GFE) and poor freezability ejaculates (PFE). In this study, sperm quality parameters of GFE and PFE, with the localization of aquaporin-3 (AQP-3) and aquaporin-7 (AQP-7) were investigated. Moreover, the relationship of AQP-3 and AQP-7 with cryotolerance of bull sperms was recorded. After fresh semen evaluation, 24 ejaculates from four Angus bulls were cryopreserved and their sperm quality parameters were analyzed by AndroVision before and after frozen-thawed. Expression of AQP-3 and AQP-7 was determined through Western blot, while their localization was detected by immunocytochemistry. The GFE and the PFE were well differentiated by sperm motility, progressive motility, viability, acrosome integrity, and mitochondrial activity, except for DNA integrity. AQP-3 was significantly higher in GFE than PFE in frozen-thawed sperm, while the AQP-7 in fresh sperm of GFE was significantly higher than that in fresh sperm of PFE and in frozen-thawed sperm of GFE (P<0.05). The location of AQP-3 was seen in the sperm mid-piece, while that of AQP-7 was recorded in the post-acrosome region, the mid and prime tailpiece. In conclusion, GFE and PGE can be well distinguished by the expression of AQP-7 in fresh semen, while AQP-7 could be a biomarker of sperm cryotolerance in bull sperm. This study is hoped to improve the efficiency of frozen sperm production and provide a reference for the improvement of frozen semen production technology in other male animals, as well as humans.