When using assisted reproductive technologies, there is seldom an evaluation of DNA integrity during sperm analysis, which is an important variable for proper embryo development. The toluidine blue staining technique allows the simultaneous evaluation of sperm chromatin and sperm head dimensions. The objectives of this study were to evaluate the applicability of the toluidine blue staining method for analyzing DNA abnormalities in epididymal sperm (from the caput, corpus, and cauda) of cats and to investigate the correlations among DNA condensation, morphology, and sperm head dimensions. The DNA alteration indexes were obtained using the toluidine blue and acridine orange techniques, and comparisons of these indexes indicated there was a 65.4% (r = 0.654; P < 0.001) correlation. The sperm from the cauda had greater chromatin stability (97.9%) than the sperm from the epididymal head (92.1%; P = 0.0023). There, however, was no difference in chromatin stability between sperm obtained from the corpus and cauda regions, indicating that these sperm were already mature. The sperm head dimension was correlated with chromatin decondensation, and the sperm head size decreased as the sperm were transported through the three epididymal regions (P < 0.0001). In addition, the percentage of sperm that were deficient in chromatin condensation decreased as the sperm were transported through the epididymal caput, corpus and cauda (26.4, 15.7, and 3.4%, respectively; P < 0.0001). Thus, the sperm head size predicts the quality of chromatin condensation in sperm cells.