To test prior results with a more sensitive technique and larger sample sizes, we assessed genetic diversity among far-flung monarch butterfly, Danaus plexippus (L.) (Lepidoptera: Nymphalidae), populations from North and South America by using mitochondrial DNA sequences. Our new data corroborate the previously documented close genetic similarity among individuals and reveal no phylogenetic structure among populations throughout the species’ New World range in North and South America. Despite this intraspecific homogeneity, the monarch is clearly distinct from its sister taxon Danaus erippus (Cramer). The evidence suggests that the monarch has colonized its current distribution in relatively recent evolutionary time. Implications for conservation and regulatory policy over interregional transfer are discussed.