We examined patterns of community structure and microhabitat requirements of Pristimantis frogs at sites of high geographic proximity in the Tambopata region of southeastern Peru. Herpetofaunal surveys were conducted at five sites encompassing approximately 131 square km of Tambopata Province, including EcoAmazonía (EA), Reserva Amazónica (RA), Explorer's Inn (EI), Sachavacayoc Centre (SC), and Tambopata Research Center (TRC). In addition to sampling and identifying all species of Pristimantis captured, environmental variables, including substrate, perch height, leaf litter depth, tree cover, and time of day, were collected and analyzed. Total frog encounters, species richness, diversity, and community similarity differed significantly among sites. Generally, similarity, richness, and diversity were not closely related to geographic proximity, but total abundance was. More individuals were captured at SC and TRC, whereas more total species were captured at TRC and EI. Diversity was the highest at TRC, followed by RA, EI, EA, and SC. Encounters of P. toftae and P. peruvianus also differed significantly among sites, with one species rarely encountered where the other was prevalent. Generally, more individuals of Pristimantis were captured during the wet season at all five sites. Significantly more individuals were captured during the night than the day. Significantly more individuals as well as species were captured on leaves and the ground versus all other substrate types, with more on leaves at night and on the ground during the day. Significantly more adults were captured higher off the ground than juveniles, with males found higher than females. There was no significant relationship between frog encounters and leaf litter or tree cover. Pristimantis peruvianus was shown to exhibit differences in microhabitat usage from P. toftae. We conclude that many factors contribute to the observed differences in diversity among the sites, requiring future studies that will also examine other environmental variables such as forest type, soil characteristics, and prey availability.