This species hides in rodent burrows and beneath leaf-litter, logs, rocks and other surface objects. It also hibernates underground or in rotting logs. In some areas it can be found in caves in, e.g., West Virginia (Green and Pauley 1987). Eggs are attached to sticks and plant stems in ponds and pools with adjacent deciduous or mixed upland forest. Presence of fishes and newts reduces reproductive success. It is not found in floodplains, swamps or marshes in New Jersey (Nyman et al. 1988). In central Pennsylvania, embryonic mortality was high in ponds below pH 4.5, though this was affected by the availability of other larval amphibians as prey (Sadinski and Dunson 1992). Courtship and egg deposition, which occurs in early spring, may occur under the ice of vernal pools. Females deposit several egg masses on sticks or emergent vegetation. The duration of egg and larval development is variable and temperature-dependent. Carnivorous larvae normally transform in July or early August and leave the pond (COSEWIC 2010). It is unclear as to whether or not it is tolerant of habitat disturbance.

This species' range was mapped by Conant and Collins (1991) as encompassing an area in the United States of America from southeastern New York through Pennsylvania and eastern and southern Ohio to southern Indiana, and southward to south-central Kentucky and northern Virginia, with an extensive area of hybridisation with Ambystoma laterale northward of this range to eastern Minnesota, northern Wisconsin, Upper Peninsula of Michigan, southern Ontario, southern Quebec, and eastward to Nova Scotia. However, for most of this range, karyological and electrophoretic data are unavailable, so the precise range of pure jeffersonianum subpopulations is uncertain (Bogart and Klemens 1997). The core of the range of pure A. jeffersonianum subpopulations likely extends from Pennsylvania southwestward to Kentucky. The jeffersonianum genome is widely distributed in eastern North America but exists primarily in hybrids (Bogart and Klemens 1997). Individuals that have solely the A. jeffersonianum genome occur in many hybridised subpopulations. Although Klemens (1993) mapped distinct ranges for A. jeffersonianum and A. laterale in Connecticut and adjacent regions, he included in the range of each species subpopulations that were dominated by the pertinent genome, including hybrids. Data presented by Bogart and Klemens (1997) indicate that the few subpopulations in New England and New York represented by only the A. jeffersonianum genome had sample sizes of only 1–3 individuals, so these actually might have been hybrid subpopulations. Phillips (1991) extended the range of A. jeffersonianum into east central Illinois, based on one juvenile raised from a larva, but since only one specimen was examined (and he did not indicate what identification criteria were used), it is unclear whether or not the subpopulation represents pure A. jeffersonianum or a hybrid subpopulation. Phillips et al. (1999) indicated the occurrence of both pure A. jeffersonianum and hybridised A. jeffersonianum ("A. platineum") in east central Illinois, and they stated that the hybrids use A. texanum sperm to activate egg development. In northern New Jersey, Nyman et al. (1988) found that triploid hybrids apparently occur wherever A. jeffersonianum is found. In Indiana and Ohio, jeffersonianum genomes exist in hybridised individuals that also contain A. texanum and/or A. tigrinum genomes (Morris and Brandon 1984, Morris 1985, Selander et al. 1993, Selander 1994).

Conservation Actions In-Place
It is uncertain if this species occurs in protected areas.

Conservation Needed
Needed conservation measures include protection of seasonal ponds and adjacent wooded areas up to at least 200–250 m from the ponds. Also, regulatory agencies should attempt to minimize forest fragmentation through habitat protection and management. 

Research Needed
Conservation goals and better taxonomic and population status information need to be developed with respect to pure and hybridized subpopulations.