Several species of mole salamanders in the genus Ambystoma are targeted by various state, provincial, and federal agencies for conservation. These salamanders have specific wetland and forested upland habitat requirements that render them vulnerable to environmental alteration. The blue-spotted salamander, Ambystoma laterale (LL) and the Jefferson salamander, A. jeffersonianum (JJ) have both been listed for protection in various parts of their ranges, but the identification of these salamanders is confusing because they often coexist with unisexual individuals that are mostly polyploid and use the sexual species as sperm donors. We used isozyme electrophoresis, blood erythrocytes, and chromosome counts in a continued effort to identify sexual and unisexual individuals in eastern North America. We examined 1377 salamanders from 118 sites in Connecticut, Massachusetts, New Jersey, New York, Pennsylvania, and Virginia. Most Pennsylvania salamanders were A. jeffersonianum (JJ) but A. laterale (LL), previously unknown from Pennsylvania, were found in that state. The two sexual species were never found together. We found diploid (LJ), triploid (LLJ; LJJ), and tetraploid (LLLJ; LJJJ; LLJJ) unisexuals. At most collecting sites, unisexuals were more numerous than sexual individuals. The association of sexual and unisexual individuals support a kleptogenic reproductive system in which the unisexuals steal genomes from their sympatric sexual sperm donors.