GST-values and its relatives (FST) belong to the most used parameters to define genetic differences between populations. Originally, they were developed for allozymes with very low number of alleles. Using highly polymorphic microsatellite markers it was often puzzling that GST-values were very low but statistically significant. In their papers, Jost (2008) and Hedrick (2005) explained that GST-values do not show genetic differentiation, and Jost suggested calculating D-values instead. Theoretical mathematical considerations are often difficult to follow; therefore, we chose an applied approach comparing two artificial populations with different number of alleles at equal frequencies and known genetic divergence. Our results show that even for more than one allele per population GST-values do not calculate population differentiation correctly; in contrast, D-values do reflect the genetic differentiation indicating that data based on GST-values need to be re-evaluated. In our approach, statistical evaluations remained similar. We provide information about the impact of different sample sizes on D-values in relation to number of alleles and genetic divergence.