The family Didelphidae can be found in a wide variety of habitats, from moist and dry tropical forests to cloud forests, mangrove swamps, grasslands, scrub, and even into temperate forests. One species, the lutrine opossum or thick-tailed opossum (Lutreolina crassicaudata) is considered to be strongly adapted to life in the South American grasslands or pampas, and readily enters lakes and streams where it swims remarkably well. Another species, the water opossum (Chironectes minimus), has as a primary habitat of streams and lakes in moist forests, making its dens in the banks.

Many species are able tree climbers and also move around on the ground; some individuals have been found high in tropical moist forest canopy trees. Other species are predominantly terrestrial, such as the members of the genera Monodelphis and Metachirus, and appear clumsy if placed in trees, even low to the ground.

Because of their long period of evolutionary isolation in South America, opossums were able to invade virtually every habitat available at every latitude and from sea level to almost 13,100 ft (4,000 m) above sea level. Most species, however, have a relatively restricted habitat and geographic range. Only species such as Didelphis virginiana and D. marsupialis can be found in several varying habitats, from temperate forests to tropical moist and dry forests to mangrove swamps.

Some species are able to exist in human-modified habitats and a few may even benefit from these, by invading banana, coffee, and citrus plantations, corn fields, and other agroe-cosystems. Other species seem particularly adept at using forest edges and secondary vegetation. Finally, many other species depend on undisturbed habitats and do not tolerate disturbance or deforestation.

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