|Abstract||Understanding the process of speciation, or the formation of new species, is a fundamental goal of evolutionary biology. Speciation requires some level of reproductive isolation between differentiating populations, regardless of the major mechanism driving the process. While allopatric speciation relies on geographic factors to maintain this barrier to gene flow, sympatric speciation lacks a geographic barrier. Sympatric speciation instead relies on ecological factors to minimize gene flow, as well as to initiate an initial speciation event within a population. Host-associated differentiation is hypothesized to be one of the primary forms of sympatric speciation, particularly for herbivorous insects. Host-associated factors may play a significant role in the host shift and speciation of Eurosta solidaginis. This gall-inducing fly has formed host races on sympatric species of goldenrod, Solidago altissima and S. gigantea. Evidence suggests that host-associated ecological factors, such as predation and competition, have played a significant role in this divergence by providing fitness differentials between the diverging populations. Such interactions have also led to further differentiation within the altissima host race, which appears to be diverging into forest and prairie populations on subspecies of S. altissima. We used microsatellite markers to analyze the extent of genetic differentiation among populations of E. solidaginis from various hosts and multiple sites in Minnesota. We found that host-associated populations of E. solidaginis on S. altissima and S. gigantea have differentiated significantly (PhiPT = 0.131; P = 0.010). Second, we found that flies collected from widely distributed populations of forest and prairie subspecies of S. altissima have also differentiated, but to a lesser extent (PhiPT = 0.066; P = 0.010). Finally, we found that differentiation among the forest and prairie altissima subpopulations varies by collection site, possibly due to ecological differences, thus producing a geographic mosaic of differentiation.