Context: Understanding habitat selection is important for determining conservation and management strategies for endangered species. The Carolina northern flying squirrel (CNFS; Glaucomys sabrinus coloratus) is an endangered subspecies found in the high-elevation montane forests of the southern Appalachians, USA. The primary use of nest boxes to monitor CNFS has provided biased information on habitat use for this subspecies, as nest boxes are typically placed in suitable denning habitat.

Aims: We conducted a radio-telemetry study on CNFS to determine home range, den site selection and habitat use at multiple spatial scales.

Methods: We radio-collared 21 CNFS in 2012 and 2014–15. We tracked squirrels to diurnal den sites and during night-time activity.

Key results: The MCP (minimum convex polygon) home range at 95% for males was 5.2 ± 1.2 ha and for females was 4.0 ± 0.7. The BRB (biased random bridge) home range at 95% for males was 10.8 ± 3.8 ha and for females was 8.3 ± 2.1. Den site (n = 81) selection occurred more frequently in montane conifer dominate forests (81.4%) vs northern hardwood forests or conifer–northern hardwood forests (9.9% and 8.7%, respectively). We assessed habitat selection using Euclidean distance-based analysis at the 2nd order and 3rd order scale. We found that squirrels were non-randomly selecting for habitat at both 2nd and 3rd order scales.

Conclusions: At both spatial scales, CNFS preferentially selected for montane conifer forests more than expected based on availability on the landscape. Squirrels selected neither for nor against northern hardwood forests, regardless of availability on the landscape. Additionally, CNFS denned in montane conifer forests more than other habitat types.

Implications: Our results highlight the importance of montane conifer to CNFS in the southern Appalachians. Management and restoration activities that increase the quality, connectivity and extent of this naturally rare forest type may be important for long-term conservation of this subspecies, especially with the impending threat of anthropogenic climate change.