Background Human activities, including changes in agricultural landscapes, often impact biodiversity through habitat fragmentation. This potentially reduces genetic exchange between previously connected populations. Using a combination of nuclear and mitochondrial markers, we investigated (i) genetic diversity and population structure at multiple spatial scales and (ii) colony genetic structure and queen mating frequency in the ant species Temnothorax nigriceps in a highly anthropized environment. Results Although the results highlighted genetic structure on a European spatial scale, they did not reveal an impact of fragmentation on a regional scale, and we did not observe any genetic population structure on a regional scale. Across all populations, regardless of their geographical location, colony structure suggested monogyny (a single queen per colony) and monandry (single mating). However, nestmates were more related than expected, indicating that large-scale dispersal does not fully prevent genetic isolation. Conclusions Despite living in fragmented patches of habitat, populations of Temnothorax nigriceps are apparently genetically not isolated at a regional scale. However, large-scale dispersal alone does not prevent genetic isolation. The ecological requirements of T. nigriceps may explain their resilience to habitat fragmentation by allowing them to survive in very small patches of suitable habitat. The deeper investigation of the diversity of functional habitats for this species should allow to appreciate better the mechanisms permitting this species to overcome the negative impacts of fragmentation.