The results of previous studies overwhelmingly suggest that RhD positive heterozygotes express better health status than Rh positive homozygous, especially in RhD negative subjects. This also applies to pregnant women. According to the Trivers-Willard hypothesis, women in better physical condition should have a male-skewed sex ratio. The aim of the present study was to test the hypothesis that RhD positive heterozygous mothers give birth to more sons than daughters. In the present cross-sectional study, we analysed data from 5,655 women who have given birth in the General University Hospital in Prague, Czech Republic between 2008-2012. Clinical records comprised maternal weight before pregnancy, number of previous deliveries, sex of the newborn, maternal RhD phenotype, and RhD phenotype of the newborn. Secondary sex ratio was significantly higher (P=0.028) in RhD positive mothers who had RhD negative newborns, i.e. in heterozygotes (SR=1.23), than in RhD positive mothers who had RhD positive newborns, i.e. in a mixed population of heterozygotes and homozygotes (SR=1.00), especially in primiparous women (P=0.013; SR=1.37 and 0.99 resp.). In line with the Trivers-Willard effect, RhD maternal heterozygous genotype is associated with male biased secondary sex ratio. The results supported the hypothesis that RhD polymorphism may be maintained due to heterozygote health advantages.