We compared the distribution of sympatric Ixodes scapularis Say and Amblyomma americanum (L.) within several suitable forested habitats at different spatial scales and characterized differences in microhabitat features accounting for the observed distribution of questing ticks. We used automatic data loggers placed in the shrub and litter layers to contrast mesoclimate and microclimate conditions experienced by questing ticks. Larger numbers of I. scapularis were collected at sites where the forest canopy was more fragmented and where the canopy contained more hardwood species than pitch pine, Pinus rigida Mill. Dominance of pine in the canopy affected the character of the shrub layer vegetation and composition of the litter layer, which concomitantly affected the microclimate conditions experienced by questing ticks. Pitch pine-dominated habitats were drier and hotter than those under a broad-leaved canopy, and questing ticks experienced increased saturation deficit in the later spring and summer in pine forest sites. The shrub layer vegetation seemed to have a moderating effect on the microclimate experienced by questing ticks and subtle differences in vegetation structure resulted in substantially different conditions as encountered by questing ticks over the space of a few meters. In contrast to questing I. scapularis, all three stages of questing A. americanum exhibited poor relationships with microclimate variables recorded in the litter and shrub layers. Further research is required to determine which environmental conditions and which habitats are most likely to support this species.