Genetic diversity levels within and between the two commercial breeding areas in the United States were analyzed using the DraI restriction fragment length polymorphism of the COI-COII mitochondrial region and 10 polymorphic microsatellite loci. The western commercial breeding population (WCBP) and the southeastern commercial breeding population (SCBP) were sampled in 1993–1994 and again in 2004–2005. The goal of this study was to characterize the genetic composition of these populations and to measure potential changes in genetic diversity and composition across the sampling period. The mitochondrial DNA haplotypes C1 and C2, characteristic of the most popular bee strains (Italians and Carniolans, respectively) sold in the United States, were the dominant haplotypes at both sample dates. The frequency of Apis mellifera mellifera M haplotypes, M4, M7, and M7′, decreased during the 10-yr span. An A1 haplotype characteristic of Africanized bees was found in the SCBP from 2005. Microsatellite analysis showed there was a loss of alleles in both the WCBP and SCBP, but the losses were not significant due to simultaneous gains of new alleles into these populations between 1993 and 2005. Genetic differences that occurred between the 1993–1994 WCBP and SCBP were still detectable in these populations sampled a decade later, suggesting that these populations could be useful sources of diversity for each other in the future.