Ecological understanding of many arboreal and semi-arboreal species is limited because of difficulties associated with studying wildlife in canopies. As part of a multi-faceted Pinus strobus canopy research project, we used camera traps located in canopies to examine diel, seasonal, and spatial patterns of Peromyscus spp.'s arboreal habitat use. From 2014 to 2018, we documented 201 events of Peromyscus spp. using the canopy over the course of 8491 camera trap nights, at three separate sites spanning a 57 km north-south transect. We detected Peromyscus spp. at heights up to 24 m, the highest above-ground observation on record, for Peromyscus leucopus (white-footed mouse) or Peromyscus maniculatus (deer mouse), the two species of Peromyscus present within our study area. To determine species-specific canopy use, we live-trapped Peromyscus at the base of each research tree and applied differential markings; right ear tags for P. leucopus and left ear tags for P. maniculatus. Camera trap images revealed both species of Peromyscus climbed with peak activity between 2000 h and 0400 h. We found ∼90% of the camera trap events occurred in Aug., Sept., and Oct. which coincides with the maturing of P. strobus cones, suggesting Peromyscus spp. may seasonally exploit arboreal habitats to forage. Daily and monthly detection probabilities for Peromyscus spp. ranged from 0.15–0.70 and from 0.14–0.91, respectively. The probability of all three cameras failing to detect an individual Peromyscus spp., given it was present in the tree, on any given day or month, ranged from 0.15–0.19 and 0.01–0.06, respectively; indicating our examination of activity patterns was robust. Our observations challenge previous ideas of the arboreal tendencies of Peromyscus spp. and further expand our understanding of the natural history of these species within the arboreal realm.