Magnolia virginiana, the type species of genus Magnolia, is a native American species belonging to section Magnolia. To better understand intraspecific taxonomy of Magnolia virginiana, we conducted molecular phylogenetic analysis based on sequences of cpDNA. Fresh leaves were collected from 28 populations (a total of 133 individuals) covering the entire distribution of the species, including the recently discovered Cuban population, and sequences of seven non-coding regions of the cpDNA were determined (ca. 5,000 bp). Based on nucleotide substitutions, ten haplotypes were recognized in M. virginiana. Phylogenetic analysis of the data matrix clearly indicated that populations of M. virginiana were divided into two major groups—one in the north and one across the south—which are essentially concordant with the morphological classification. Five nucleotide substitutions were found between them. Within the southern group, one common haplotype widely distributed, and populations of Texas (and adjacent areas) and western Tennessee showed a unique haplotype with an additional substitution(s), respectively. Less common haplotypes were found in Florida. The haplotype of the Cuban population was the same as the common haplotype of the southern group.