The Jefferson complex comprises the Jefferson Salamander (Ambystoma jeffersonianum) and the Blue-spotted Salamander (Ambystoma laterale), in addition to unisexual salamanders, which present various combinations of the nuclear genome of the two bisexual species and show different levels of ploidy. The unisexuals arose by an ancestral hybridization event, and they all share a similar mitochondrial haplotype clearly different from that of the bisexual species. Although adults of A. jeffersonianum and A. laterale are usually easily differentiated morphologically, unisexuals can be difficult to identify visually because they may possess intermediate characters, or morphological traits from either of their bisexual counterparts. In the present study, we introduce a novel way to discriminate between bisexual and unisexual salamanders of the Jefferson complex based on taxon-specific primers designed in the mitochondrial cytochrome b region. This molecular approach allows for a simple, rapid, and non-invasive identification of unisexuals, using a single polymerase chain reaction (PCR) and small tissue samples that can easily be obtained from live specimens. We believe that this approach will be useful to screen a large number of individuals quickly in order to identify populations of the Jefferson Salamander, a crucial step towards the conservation of this threatened species.