Sperm lipids are important for sperm viability, maturity and function. This study aimed to identify cholesterol and desmosterol composition of human spermatozoa of two sperm populations separated on Sil‐Select gradient. Forty‐eight males were divided into four groups namely healthy men (n = 13), asthenozoospermia (n = 11), asthenoteratozoospermia (n = 10) and oligoasthenoteratozoospermia (n = 14). Sperm cholesterol and desmosterol were estimated in two human sperm population separated by centrifugation in a discontinuous Sil‐Select gradient. The results showed that cholesterol and desmosterol were the major sterols in human spermatozoa. Spermatozoa recovered from upper/lower layer interface (fraction I) had low fertilization potential, while those from the base (fraction II) had high fertilization potential. Median values of cholesterol and desmosterol in fraction I were 2.55 μmol and 0.77 μmol/10⁹ spermatozoa and in fraction II were 1.16 μmol and 0.27 μmol/10⁹ spermatozoa. Cholesterol/desmosterol ratio was significantly higher in fraction II than I (4.8 vs. 3.2, p < 0.01). Cholesterol, desmosterol, total phospholipids and sterols/phospholipids were negatively correlated with sperm concentrations, sperm motility, linear velocity, normal sperm morphology and acrosome reaction percentage whereas cholesterol/desmosterol ratio was positively correlated with these parameters. It is concluded that the difference in sterol composition of sperm subpopulations separated on Sil‐Select gradient suggests that composition of sterols is related to sperm functions.