There is growing interest in the use of single nucleotide polymorphisms for evolutionary and population genetics. We tested the efficacy of one of the available single nucleotide polymorphism techniques, single-base extension, in distinguishing four cryptic species of Microtus. Sequence data were available for these species at nuclear and mitochondrial loci and their identity could be independently confirmed using karyotypes. We found that the development and optimization of single nucleotide polymorphisms required extensive effort, and that the method accurately identified the correct nucleotide at single nucleotide polymorphism sites ≈ 90% of the time at the conserved nuclear locus. Correct identification rates were much lower at the highly variable mitochondrial locus.