The soybean aphid, Aphis glycines Matsumura, is a recent invasive pest of soybean in North America. Currently, much research is focused on developing and characterizing soybean cultivars expressing host-plant resistance. During the initial phases of host-plant resistance screening, many of these studies use soybean aphid laboratory populations. Previous studies in other systems have documented substantial differences among laboratory and field populations. Whether or not this pattern exists in A. glycines is unknown, but it is extremely important when estimating the level of selection and virulence to host-plant resistant soybeans. In this study, we used seven microsatellite markers to estimate and compare genetic diversity and differentiation among five laboratory and 12 field populations. Our results indicate that soybean aphid laboratory populations are severely lacking in genotypic diversity and show extreme genetic differentiation among each other and to field populations. Continued use of laboratory populations for initial soybean aphid resistance screening could lead to erroneous estimations of the potential success for host-plant resistance.