Knowledge of the habitat and spatial characteristics of natal dens used by female river otters (Lontra canadensis) is limited to a few observations. Documentation of these characteristics in human-altered landscapes may improve management opportunities for this species. We monitored 8 adult (>2 y old) radio-marked female otters during the natal denning season (March–May) in southeastern Minnesota during 2003–2004 and quantified 10 micro- and 2 macro-habitat characteristics of dens. Females began denning in March, with a mean initiation date of 31 March, and used natal dens for a mean of 49 d (se = 3). Two females used man-made brush piles as dens, four used small limestone caves, one used a cavity in the roots of a big-toothed aspen (Populus grandidentata) and one used a beaver (Castor canadensis) bank-den. Dens were located a mean of 316 m (se = 79) from the nearest body of water and averaged 61 m (se = 15) of elevation higher than the nearest body of water. Seven of eight females placed dens outside of their normal activity areas, and all females appeared to select den sites that were protected from flood events. Our results suggest that to promote successful reproduction of river otter populations, managers should protect potential denning sites in upland habitats adjacent to bodies of water.