The Least brook lamprey (LBL), Lampetra aepyptera, is a nonparasitic fish species native to the eastern United States found in small streams in the Mississippi, Mobile Basin, and Atlantic slope drainage systems. The focus of this project is to identify the geographic pattern of mitochondrial DNA variation and to recognize phylogenetically distinct clades within this species. I investigated the phylogeography of the LBL by sequencing 351 base pairs of the mitochondrial ND3 gene and approximately 650 bases of the mitochondrial control region. Samples from 21 populations throughout the LBL’s distribution were sequenced. The combined ND3 and control region data sets identified 23 unique haplotypes. Ten highly differentiated clades detected correspond to drainage systems and/or geographic location. My data suggest a rapid diversification during the late Miocene and/or Pliocene, and that the LBL consists of several undescribed species. LBL distribution is likely the result of vicariance, dispersal and isolation events.