Background: The flea beetle Phyllotreta nemorum is polymorphic for resistance to the defence of the atypical host plant Barbarea vulgaris G-type. We investigated the factors influencing the geographical distribution of this resistance in a structured population in Denmark, where the proportion of flea beetles resistant to the defences of B. vulgaris is remarkably low for individuals collected on other host plants than B. vulgaris. We tested the hypothesis that the resistance trait is selected against when beetles live on non-defended host plants that are geographically separated from a patch of defended host plants. New primers were developed for 11 polymorphic microsatellite loci of Phyllotreta nemorum which, in addition to 10 previously developed microsatellite markers, were used to assess the genetic population structure nearby Kværkeby. Results: Outlier detection methods were applied to different measures of pairwise genetic differentiation between samples at neutral behaving microsatellite loci as well as the resistance trait and yielded the resistance trait as a candidate subject to selection. This also applied to the comparison between flea beetle subpopulations on non-defended host plants at different geographical distances from a patch of B. vulgaris G-type plants. Conclusions: These results suggest that the different frequencies of flea beetle resistance on patches of non-defended host plants cannot be explained by mere dispersal limitations, but that a locus-specific effect, such as directional selection, must also play a role. The possible mechanism of such selection is discussed, along with the comparison of different genetic differentiation measures and mutation models in population genomic studies.