Habitat loss and resultant fragmentation are major threats to biodiversity, particularly in tropical and subtropical ecosystems. It is increasingly urgent to understand fragmentation effects, which are often complex and vary across taxa, time and space. We determined whether recent fragmentation of Atlantic forest is causing population subdivision in a widespread and important Neotropical seed disperser: Artibeus lituratus (Chiroptera: Phyllostomidae). Genetic structure within highly fragmented forest in Paraguay was compared to that in mostly contiguous forest in neighbouring Misiones, Argentina. Further, observed genetic structure across the fragmented landscape was compared with expected levels of structure for similar time spans in realistic simulated landscapes under different degrees of reduction in gene flow. If fragmentation significantly reduced successful dispersal, greater population differentiation and stronger isolation by distance would be expected in the fragmented than in the continuous landscape, and genetic structure in the fragmented landscape should be similar to structure for simulated landscapes where dispersal had been substantially reduced. Instead, little genetic differentiation was observed, and no significant correlation was found between genetic and geographic distance in fragmented or continuous landscapes. Furthermore, comparison of empirical and simulated landscapes indicated empirical results were consistent with regular long-distance dispersal and high migration rates. Our results suggest maintenance of high gene flow for this relatively mobile and generalist species, which could be preventing or significantly delaying reduction in population connectivity in fragmented habitat. Our conclusions apply to A. lituratus in Interior Atlantic Forest, and do not contradict broad evidence that habitat fragmentation is contributing to extinction of populations and species, and poses a threat to biodiversity worldwide.