The structure and genetic diversity of a widely distributed species in a recently colonized area is influenced by the colonizing lineages, life-history traits, and biotic and abiotic factors. The connection established during the Pliocene between North and South America allowed the nine-banded armadillo (Dasypus novemcinctus) to expand its distributional range northward. High levels of genetic diversity have been recorded in South America, whereas low levels have been detected in populations in the United States, perhaps due to a founder effect during colonization. By sampling animals from Mexico and a few other areas, we test the hypothesis that armadillos in North America were derived from a single founding lineage, and assess whether this newly colonized region shows demographic signatures of expansion. We sequenced the mitochondrial control region of 157 individuals and genotyped microsatellites of 116 individuals. Our mitochondrial results showed 2 divergent lineages with high genetic variation in Mexico when compared to United States populations, suggesting that this species has a higher effective population size in Mexico. Samples from Central and South America indicate that both lineages differentiated prior to their arrival in Mexico. Lineages showed a historical demographic expansion, due probably to the large area of colonization. Clear genetic structure was observed with mitochondrial DNA, whereas microsatellites showed low levels of genetic differentiation. This contrasting pattern can be caused by male-biased dispersal. We conclude that North American populations of D. novemcinctus are derived from 2 founding lineages and show the consequences of the Great American Biotic Interchange influencing genetic patterns in the nine-banded armadillo in Mexico.