Technical Abstract: Heteroecious holocyclic aphids alternate between sexual and asexual reproduction on primary and secondary hosts, respectively. Most of these aphids are generalists, but the aphid specialist Aphis glycines survives only on the primary host buckthorn (Rhamnus spp.) and the secondary host soybean (Glycine max). Due to this specialization and sparse primary host distribution, colonization events could be localized and involve founder effects, impacting genetic diversity, population structure and adaptation. We characterized changes in genetic diversity and structure across time among A. glycines populations. Populations were sampled from secondary hosts twice in the same geographic location: once after secondary colonization (early-season), and again immediately prior to primary host colonization (late-season). We specifically tested for evidence of founder effects and genetic isolation in early-season populations, and whether or not late-season dispersal restored genetic diversity and reduced fragmentation. A total of 24 SNPs and 6 microsatellites were used to generate and compare population genetic statistics including the number of genotypes, FST, and spatial autocorrelation. We found significantly lower levels of genetic diversity and higher levels of genetic isolation among early-season collections, indicating secondary host colonization occurred locally and involved founder effects. Pairwise FST decreased from 0.046 to 0.017 in early and late collections, respectively, and while genetic relatedness significantly decreased with geographic distance in early-season collections, no spatial structure was observed in late-season collections. Thus, late-season dispersal counteracts the population genetic impacts of secondary host colonization through homogenization and increases genetic diversity prior to primary host colonization.