OBJECTIVE To investigate variation of sperm DNA fragmentation index (DFI) in male partners of infertile couples. METHODS A total of 539 males between April 2009 and April 2012 were analyzed. At least one repeated routine semen analysis and sperm DNA fragmentation test were performed for each sample by sperm chromatin dispersion (SCD) analysis following World Health Organization guidelines. Coefficient of variation (CV) for DFI was calculated. RESULTS Respectively, 1, 2, 3 and 4 repeated SCD analyses were carried out on 473, 59, 6 and 1 semen samples. The median interval between the first and repeated SCD measurements was 3.0 (1.0-11.0) months. For the first tested samples, the between-sample coefficient of variation (CVB) for DFI was 71.2%. A significant difference has been found between DFI of the first measurement and DFI of repeated measurement in 0.5 to 3 months, 3 to 12 months and 12 to 34 months (P< 0.01). Compared with the first test, 26.3% of males were on both sides of the cut-off point of 18%. The median within-subject coefficient of variation (CVw) for DFI of 539 men was 26.0% (12.6%-42.8%). And the median CVw DFI was significantly lower compared with CVw of sperm count, concentration, progressive motility and normal morphology (P< 0.01). Significant correlations were found between the CVw DFI and sperm count, concentration and interval among the samples (P< 0.05). CONCLUSIONS DFI of male partners for infertile couples is a parameter with substantial variation, repeated SCD measurements are therefore recommended.