The Eurycea bislineata complex (“two-lined salamanders”) of eastern North America contains six described species, of which three have very similar morphologies and relatively broad geographic distributions, and three have more divergent morphologies with narrow geographic distributions. Recent molecular phylogenetic analyses found that four of the six species in the complex contain deep genetic structure, and that two of the species (E. cirrigera and E. wilderae) are paraphyletic in mitochondrial and nuclear DNA, inferring that current taxonomy does not reflect actual species boundaries in the complex. The member of this complex in the Sandhills physiographic region of south-central North Carolina, USA, is notable for its distinctive coloration and ecology, and a study on allozymic variation published over 30 years ago demonstrated that it is genetically distinct but clustered within the paraphyletic E. cirrigera. This study investigates the taxonomic status of the Sandhills population using morphology, mitochondrial DNA, and a 21-locus nuclear DNA data set to test if the Sandhills taxon represents a local ecomorph that is conspecific with adjacent populations of E. cirrigera, or if it represents a divergent evolutionary lineage that warrants taxonomic recognition. Mitochondrial and nuclear DNA revealed that the Sandhills taxon is genetically distinct and phylogenetically unrelated to adjacent populations of E. cirrigera. Principal components analysis of 316 adult specimens of the Sandhills taxon and the three morphologically similar species in the complex found considerable overlap among these taxa, but pairwise comparisons of heavily loading morphological characters showed that the Sandhills taxon usually has a shorter body, shorter tail, and narrower head. Based on these corroborated lines of evidence, the hypothesis that the Sandhills taxon represents only a local ecomorph is rejected and it is described as a new species. Clarification of the extent of its geographic range (including its possible presence in South Carolina), differentiation of larvae from other members of the complex, and verification of hypothesized narrow zones of hybridization with E. cirrigera at the peripheries of its geographic range are needed. The description of the Sandhills taxon brings the number of endemic salamander species in North Carolina to seven. Integrative taxonomic revisions of the E. bislineata complex, particularly E. cirrigera and E. wilderae, are needed to estimate species diversity and distributions of these salamanders more accurately.

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