Adult Asian long-horned beetles, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae: Lamiinae), were discovered in Ontario, Canada, in 2003 in the vicinity of a commercial warehouse. Trees were heavily scarred with signs of attack and larvae and adult beetles were common, suggesting that there had been multiple generations at the site. We amplified 16 microsatellite loci from 326 beetles to examine genetic diversity in this population. Based on Hardy—Weinberg equilibrium, 6 of 16 loci were monomorphic and 8 were not, indicating nonrandom mating. Measures of microsatellite genetic diversity and mitochondrial DNA haplotype diversity were significantly lower than those in A. glabripennis from China and Korea but were not significantly different from those in the New York City population. The proportion of different multilocus genotypes in the Ontario population was lower than in the populations in New York City and Linden, New Jersey. These results suggest that limited genetic diversity in the Ontario population has not hampered reproduction of this invasive insect. This genetic signature is common in other invasive species, likely because a population is founded by a few closely related individuals, or a large founding population suffers subsequent genetic bottlenecks.