Estimating the extent of genetic differentiation between populations is an important measure in population genetics, ecology and evolutionary biology. Fixation index or FST is an important measure, which is routinely used to quantify this. Previous studies have shown that FST estimated for selectively constrained regions was significantly lower than that estimated for neutral regions. By deriving the theoretical relationship between FST at neutral and constrained sites we show that an excess in the fraction of deleterious variations segregating within populations compared to that segregates between populations is the cause for the reduction in FST estimated at constrained sites. Using whole genome data, our results revealed that the magnitude of reduction in FST estimates obtained for selectively constrained regions was much higher for distantly related populations compared to those estimated for closely related pairs. For example, the reduction was 47% for comparison between Europeans-Africans, 30% for European-Asian comparison, 16% for the Northern-Southern European pair and only 4% for the comparison involving two Southern European (Italian and Spanish) populations. Since deleterious variants are purged over time due to purifying selection, their contribution to the among-population diversity at constrained sites decreases with the increase in the divergence between populations. However, within-population diversity remains the same for all pairs compared and therefore FST estimated at constrained sites for distantly related populations are much smaller than those estimated for closely related populations. Our results suggest that the level of population divergence should be considered when comparing constrained site FST estimates from different pairs of populations.