The Streaked Horned Lark (STHL; Eremophila alpestris strigata) is a federal candidate for listing under the Endangered Species Act. We evaluated the conservation status and level of genetic diversity of the STHL using the complete mitochondrial ND2 gene. We sampled 32 STHLs from the southern Puget Sound region, the Pacific coast, and Whites Island in the Columbia River of Washington, and additional 68 horned larks from Alaska, alpine and eastern Washington, Oregon, California, and Asia (outgroups). Our Maximum Likelihood analysis of 32 haplotypes identified three geographically concordant clades in Pacific coast states: Pacific Northwest (alpine and eastern Washington, Alaska), Pacific Coast (western Washington, California), and Great Basin (eastern Oregon). Each of the three clades was supported by bootstrap values ‡86%. The distance among them varied from 0.72 to 0.79% nucleotide divergence excluding intraclade variation. The relationship among the clades was not resolved. AMOVA also showed significant structuring of haplotypes among the three clades. Differences among clades accounted for 75.7% of sequence variation, differences among localities within clades accounted for 12.1%, and differences among individuals within localities accounted for the remaining 12.2%. Although STHL populations were closely related to the Californian sample, they appeared unique and isolated. All pairwise Fst values involving the STHL samples were significant (except between themselves). STHLs appear to have remarkably low genetic diversity; all 32 STHLs shared the same haplotype. Even with small sample sizes, all other localities had multiple haplotypes. Because the STHL appears to be unique and isolated, and to have little genetic diversity our data suggest it should be a conservation priority.