BACKGROUND Telomeres are particular sequences of DNA located at the end of the eukaryotic chromosomes that are essential for genome integrity. Telomere length in spermatozoa differs among males, as well as spermatozoa. Also, decreased telomere length in spermatozoa of infertile men is associated with the reduction of fertility potential and embryo quality. Density gradient centrifugation (DGC) and swim-up are useful techniques for separation of spermatozoa with longer telomeres. Also, the selection of sperm based on surface negative electric charge or "Zeta potential", can separate high percentage of spermatozoa with intact chromatin compared to DGC alone, and also the combination of DGC-Zeta can improve clinical outcomes of infertile men candidate for intracytoplasmic sperm injection (ICSI). Therefore, we compared sperm telomere length and DNA fragmentation between two sperm preparation procedures, namely DGC and zeta potential. METHODS In this experimental study, we assessed sperm telomere length and DNA fragmentation by quantitative real-time polymerase chain reaction (PCR) and TUNEL assay methods, respectively. The spermatozoa were obtained from infertile men with normozoospermia between September 2017 and December 2017 and prepared either by DGC or zeta potential methods. Sperm telomere length was expressed as relative and absolute units. RESULTS Compared with washed semen samples or control, no significant (P>0.05) difference was observed in the mean relative or absolute sperm telomere length when the two methods DGC or zeta potential were compared. However, the mean percentage of DNA fragmentation was significantly (P<0.05) lower in spermatozoa prepared by DGC or zeta potential methods than spermatozoa obtained from control samples. CONCLUSIONS This is the first study that compared the effect of DGC and zeta potential as the sperm preparation methods on sperm telomere length. It seems that both methods can select sperm population with high DNA integrity and the same sperm telomeres length.