There is insufficient evidence on correlation between DNA fragmentation and recurrent implantation failure (RIF) to introduce sperm DNA testing as a part of the clinical practice. Thus, this study investigated the influence of sperm DNA fragmentation on the incidence of RIF. Partners of 110 women with RIF following intracytoplasmic sperm injection (ICSI), and 50 recent fathers (control) were examined. Sperm DNA fragmentation was examined by the sperm chromatin dispersion (SCD) test, Aniline blue test (AB), Acridine orange (AO) test and DNA diffusion assay. The study also examined different aspects causing DNA fragmentation such as oxidative stress by biochemical measuring of reduced glutathione (GSH) and malondialdehyde (MDA) levels, and the reactive oxygen species (ROS) level was also studied using dichlorofluorescin diacetate. There were no obvious differences in sperm DNA fragmentation index measured among different used tests. However, AB was found to provide clear and stable clinical cut-off levels, and considered as the most easy and inexpensive assay. Therefore, AB can be recommended for a clinical sperm DNA damage evaluation as a routine test. Moreover, impaired chromatin packaging, apoptosis and ROS result in significant DNA damage in human spermatozoa of RIF group compared to control and positive pregnancy groups.These results support the hypothesis that sperm DNA fragmentation is an important cause of RIF, and that sperm DNA integrity testing has value as a diagnostic tool in investigation and treatment of infertility.