We examined the phylogeography of the Least Brook Lamprey (Lampetra aepyptera) to recognize phylogenetically distinct clades within this species and to estimate the relative contributions of vicariance and dispersal to the distribution of genetic variation. A combined data set from mitochondrial ND3 and control region sequences identified 23 unique haplotypes among 21 populations. Eleven highly differentiated clades within L. aepyptera were detected. Their distributions correspond to different drainages and/or locations within drainage. Clades differed by an average of 4.5% (range 2.5–9.0%) sequence divergence. Only two haplotypes were shared among any populations. Considerable differentiation among adjacent drainages and limited sharing of haplotypes suggested little historical or contemporary gene flow. Although clades were highly differentiated, resolution of clade relationships was limited. The presence of highly differentiated clades in different drainages suggests that vicariance has had a profound effect on the distribution of genetic variation in L. aepyptera. The lack of resolution of clade relationships within this species suggested a single event that simultaneously isolated many of these populations. Rising sea levels causing a marine incursion into the Mississippi embayment during the Pliocene could have been such an event.