The successful implementation of ICSI has provided a unique means of allowing couples suffering from severe male infertility to achieve their reproductive goals. However, despite the great therapeutic advantages of the technique, ICSI often provides solutions to clinicians in the absence of an aetiological or pathophysiological diagnosis. The development of a sequential diagnostic schedule for patients consulting for fertility disturbances would be an ideal method of approach. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology and chromatin staining among fertile and subfertile men. Both chromomycin A3 (CMA3) and acidic aniline blue (AAB) were employed to record chromatin packaging quality among 58 men visiting the andrology laboratory. Intra- and interassay variations were initially recorded for fertile sperm donors. The coefficients of variation (CV) for all intra- and inter-assay assessments were < 12%. Chromatin packaging was significantly and negatively correlated with normal sperm morphology, namely r = 0.40 (P = 0.001) and r = 0.33 (P = 0.001) for CMA3 and AAB, respectively. Receiver operator characteristics illustrated sensitivity and specificity values of 75% and 82% for CMA3 and 60% and 91% for AAB, respectively. Significantly different CMA3 and AAB staining was recorded among men with severe teratozoospermia (< 4% normal forms) when compared with normozoospermic men (> 14% normal forms), namely 49% vs. 29% for CMA3 and 51% vs. 26% for AAB staining, respectively. Chromatin packaging assessments should be a valuable addition to the sequential diagnostic programme in an assisted reproduction arena.