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We examined mitochondrial DNA (mtDNA) variation in six Mennonite communities from Kansas (Goessel, Lone Tree, Garden View, Meridian, and Garden City) and Nebraska (Henderson) to determine their genetic structure and its relationship to population history. Mitochondrial DNA haplogroup and haplotype information were obtained from blood samples from 118 individuals. Molecular genetic variation was analyzed using diversity measures, neutrality test statistics, spatial analysis of molecular variance (SAMOVA), and multidimensional scaling plots. The Mennonite samples exhibited eight western European mtDNA haplogroups: H, HV0, I, J, K, T, U, and X. Comparable to other populations of European descent, haplogroup H was the most frequent in all six communities and ranged from 35% in Lone Tree to 75% in Old Order Mennonites from Garden City. Fifty-eight different mtDNA haplotypes were found in these groups with only one shared among all six populations. Haplotype diversities varied from 0.81 in Goessel to 0.96 in Henderson and Garden View. Multivariate statistical analysis of these populations indicates that these Anabaptist communities formed new congregations by fissioning along familial lines. Population subdivision of these communities into congregations supports previously documented patterns of fission-fusion. These haploid molecular data provide a more accurate reflection of biological relationships between midwestern Mennonite communities than evidence based on classical genetic markers.