The wasp spider Argiope bruennichi (Scopoli, 1772) is of Mediterranean-Pontian origin, but for decades it has been expanding northwards, including into the territory of Poland. Based on well-documented expansion records, we can distinguish “old” (south-eastern and south-western) and “new” populations (north-eastern), respectively, from the 1930s to the 2000s. In Poland, some populations of A. bruennichi were expected to be more genetically isolated from others, due to distance effects or differential times of arrival. We evaluated whether the oldest populations were in a state of Hardy-Weinberg equilibrium (HWE), and whether recently founded populations were in an expansion phase. Specimens of A. bruennichi (n = 184) were collected at six localities in Poland and single sampling sites in Italy and Japan. Nine microsatellite loci were amplified although only five were useful in the final analyses. Based on the genotypes obtained, we estimated basic measures of genetic diversity and tested for deviation from HWE. The results showed a low level of polymorphism amongst the investigated markers, and accordingly, we found a low genetic diversity in populations. Only populations from Italy and Japan, and one population from Poland, were in HWE. The level of genetic differentiation among sampling sites from Poland was also very low. The high dispersal ability of the wasp spider appears to have facilitated high gene flow among populations. The peripheral and recently settled populations were characterized by the highest heterozygosity and the lowest inbreeding coefficient (F_IS)_. The remaining Polish populations are therefore still in the expansion phase, as indicated by deviations from HWE.