Two of the four members of subsection Contortae of the genus Pinus occur in the southeastern United States: Pinus virginiana, which ranges throughout the southern and central Appalachian Mountains, and P. clausa, which is restricted to Florida and southern Alabama. We examined allozyme variation within P. virginiana and genetic relationships between this species and the two varieties of P. clausa (var. clausa and var. immuginata). P. virginiana maintains more genetic diversity at both the species (Hes = 0.139) and population (Hep = 0.128) levels than the other three species in the subsection, which may reflect the combination of its widespread distribution and the absence of cone serotiny. Genetic differentiation among populations in P. virginiana was relatively low (GST = 0.053), but significant contrasts in allozyme frequencies and genetic diversity were apparent between populations to the northwest vs. outheast of the Appalachian Mountains. These regional differences likely resulted initially from historical processes that occurred during the Pleistocene and early Holocene, and have been reinforced by modern selective pressures and barriers to gene flow. The mean genetic distance between populations of P. virginiana and P. clausa (D = 0.071) was greater than that between populations of the two varieties of P. clausa (D = 0.012), which suggests that the two varieties diverged at some point after the separation of the two species.