Studies of herbivorous insects have played a major role in understanding how ecological divergence can facilitate genetic differentiation. In contrast to the majority of herbivorous insects, grasshoppers as a group are largely polyphagous. Due to this relative lack of intimate grasshopper-plant associations, grasshopper-plant systems have not played a large part in the study of host-associated genetic differentiation. The oligophagous grasshopper, Hesperotettix viridis (Thomas), is endemic to North America and feeds on composites (Asteraceae) within the tribe Astereae. Previous work has shown both preference and performance differences between H. viridis individuals feeding on either Solidago mollis or Gutierrezia sarothrae. Using 222 AFLP markers, we examined the genetic relationships among 38 H. viridis individuals feeding on these plants both in sympatry and allopatry. Neighbour-joining analysis resulted in two distinct host-associated clades with 71% bootstrap support for host-associated monophyly. Analyses of molecular variation (amova) revealed significant genetic structuring with host plant accounting for 20% of the total genetic variance while locality accounted for 0%. Significant genetic differentiation was detected between S. mollis-feeders and G. sarothrae-feeders even when the two were present at the same locality. These results are consistent with observed differences in preference and performance between H. viridis grasshoppers feeding on either G. sarothrae or S. mollis and indicate that H. viridis is comprised of at least two genetically distinct host plant-associated lineages.