Genetic diversity and gene flow were examined within and among populations of nine-banded armadillos (Dasypus novemcinctus) from Paraguay. Although D. novemcinctus is hunted regularly and constitutes an important protein resource in Paraguay, no conservation or management program exists for this species. To contribute toward conservation planning for this species, we examined intra- and interpopulation DNA sequence variation in the mitochondrial cytochrome-b gene from 75 specimens representing 5 populations from Paraguay. Twenty-five haplotypes were identified among the 75 individuals examined. Haplotype diversity (h) and nucleotide diversity (π) ranged from 0.68 to 0.94 and 0.003 to 0.005, respectively. Although genetic homogeneity was observed among all 5 populations, nested-clade analysis revealed continuous range expansion, isolation by distance, and long-distance dispersal of female nine-banded armadillos.