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The aim of this study was to evaluate the effect of sperm chromatin anomalies on fertilization outcome post‐intracytoplasmic sperm injection (ICSI). Therefore, along with semen parameters, Chromomycin A3 (CMA3) staining for protamine deficiency, aniline blue staining for excessive histones, SDS for sperm chromatin stability and SDS + EDTA for the ability of sperm to undergo decondensation were carried out on 55 semen samples from patients referred to the Isfahan Fertility and Infertility Center for ICSI. The results showed that among the aforementioned tests and semen parameters only CMA3 showed a significant correlation with fertilization outcome post‐ICSI. Patients were also grouped according to CMA3 level of <30% or >30% or fertilization rate of <50% or >50%. The results show that the mean percentage fertilization and mean percentage of CMA3 positivity is different in both groups, respectively. The …