The geographical range of unisexual Ambystoma overlaps with four bisexual species that also breed in spring ponds. Several of these species are of conservation concern, and both adults and larvae can be difficult to distinguish morphologically from unisexuals.

Here we present a rapid molecular method for screening unisexuals, whose mtDNA is most similar to Ambystoma barbouri. A 258 bp segment of the cytochrome b gene was amplified in six Ambystoma species and exemplar unisexuals by PCR using taxon-specific primers. An internal 113 bp segment was amplified only in unisexuals and A. barbouri using Universal forward and Hybrid reverse primers. Multisequence alignment comparing the nucleotide sequence where Hybrid reverse primer anneals revealed nucleotide diversity in this region among Ambystoma species.

This simple method for discriminating between unisexuals and bisexuals, excluding A. barbouri, can be applied prior to further research on these declining species.