Fetal heart rate (FHR) variability throughout gestation reflects aspects of fetal development, and its analysis has been used for the assessment of fetal well-being.The objective of this study was to provide a gender-specific analysis of the evolution of FHR variability indices throughout gestation, using linear time-domain, spectral and nonlinear FHR indices.A large retrospective study was carried out using antepartum FHR recordings obtained from 4713 male and 4110 female fetuses, with normal pregnancy outcome, between 2004 and 2013, with gestational ages ranging between 25 and 40weeks.FHR variability was analysed through linear time-domain methods, as well as using spectral analysis and entropy indices. Evolution of FHR indices throughout gestation was analysed through Spearman correlation coefficient. Comparison between male and female fetuses was performed using nonparametric bootstrap 95% confidence intervals for the median.Mean FHR decreased significantly throughout gestation, whereas most variability indices increased. Sympatho-vagal balance measured by spectral analysis exhibited two local maxima at 29–30 and 34–35weeks and decreased afterwards. Entropy indices increased until around the 34th week, slightly decreasing after the 37th week. Female fetuses presented higher mean FHR and entropy from the 34th week afterwards, and lower short-term variability and sympatho-vagal balance in the same period.Spectral and entropy analysis should be considered as a complement to conventional FHR variability analysis, aiming at a better characterization and follow-up of fetal development/maturation throughout gestation. Additionally, gestational age needs to be considered when defining reference ranges for FHR indices in systems of computerized analysis.