Many polyphagous herbivore insects considered as a single species are indeed comprised of genetically differentiated host related races that utilize several unrelated plant families. Aphis gossypii Glover, one of the extremely polyphagous aphid species, has more than 100 host plants distributed worldwide. In Korea, it takes holocyclic life cycle, with a number of primary hosts, which is unusual for other aphid species. However, former population genetic studies on A. gossypii have dealt with the populations from only a few agricultural host plants. In this study, we hypothesized that different primary host utilization may be restricted for certain host races and this may play a key role of the genetic differentiation in this species. We collected the cotten aphid populations from 36 different host plants, 5 primary and 31 secondary hosts including various wild and non-cultivated plants. To determine population genetic structure, we analyzed genetic differentiation among those populations using 9 microsatellite loci. As results, four genetically distinct clusters were verified. Of the five primary host populations, there were significant gaps in genetic differences between Rhamnus-associated group and Hibiscus-associated group, and the other populations were affinitive with the Hibiscus-associated group. Overall, primary host populations have a higher level of genetic diversity than secondary host populations. Several populations associated with secondary hosts such as spiderwort, madder, and Korean lettuce were considerably differentiated in the result of principle component analysis, which nearly showed the species level difference similar to Aphis glycines Matsumura and Aphis sp. ex Rhamnus. Thus, there could be reproductively isolated by the loss of primary hosts, and might be already speciated from A. gossypii