Effects of habitat fragmentation on genetic diversity vary among species. This may be attributed to the interacting effects of species traits and landscape structure. While widely distributed and abundant species are often considered less susceptible to fragmentation, this may be different if they are small sized and show limited dispersal. Under intensive land use, habitat fragmentation may reach thresholds at which gene flow among populations of small-sized and dispersal-limited species becomes disrupted. Here, we studied the genetic diversity of two abundant and widespread bush crickets along a gradient of habitat fragmentation in an agricultural landscape. We applied traditional (GST, θ) and recently developed (, D) estimators of genetic differentiation on microsatellite data from each of twelve populations of the grassland species Metrioptera roeselii and the forest-edge species Pholidoptera griseoaptera to identify thresholds of habitat fragmentation below which genetic population structure is affected. Whereas the grassland species exhibited a uniform genetic structuring (GST = 0.020–0.033; D = 0.085–0.149) along the whole fragmentation gradient, the forest-edge species’ genetic differentiation increased significantly from D < 0.063 (GST < 0.018) to D = 0.166 (GST = 0.074), once the amount of suitable habitat dropped below a threshold of 20% and its proximity decreased substantially at the landscape scale. The influence of fragmentation on genetic differentiation was qualitatively unaffected by the choice of estimators of genetic differentiation but quantitatively underestimated by the traditional estimators. These results indicate that even for widespread species in modern agricultural landscapes fragmentation thresholds exist at which gene flow among suitable habitat patches becomes restricted.