Antibiotic treatment can impact nontarget microbes, enriching the pool of resistance genes available to pathogens and altering community profiles of microbes beneficial to hosts. The gut microbiota of adult honeybees, a distinctive community dominated by eight bacterial species, provides an opportunity to examine evolutionary responses to long-term treatment with a single antibiotic. For decades, American beekeepers have routinely treated colonies with oxytetracycline for control of larval pathogens. Using a functional metagenomic screen of bacteria from Maryland bees, we detected a high incidence of tetracycline/oxytetracycline resistance. This resistance is attributable to known resistance loci for which nucleotide sequences and flanking mobility genes were nearly identical to those from human pathogens and from bacteria associated with farm animals. Surveys using diagnostic PCR and sequencing revealed that gut bacteria of honeybees from diverse localities in the United States harbor eight tetracycline resistance loci, including efflux pump genes (tetB, tetC, tetD, tetH, tetL, and tetY) and ribosome protection genes (tetM and tetW), often at high frequencies. Isolates of gut bacteria from Connecticut bees display high levels of tetracycline resistance. Resistance genes were ubiquitous in American samples, though rare in colonies unexposed for 25 years. In contrast, only three resistance loci, at low frequencies, occurred in samples from countries not using antibiotics in beekeeping and samples from wild bumblebees. Thus, long-term antibiotic treatment has caused the bee gut microbiota to accumulate resistance genes, drawn from a widespread pool of highly mobile loci characterized from pathogens and agricultural sites. We found that 50 years of using antibiotics in beekeeping in the United States has resulted in extensive tetracycline resistance in the gut microbiota. These bacteria, which form a distinctive community present in healthy honeybees worldwide, may function in protecting bees from disease and in providing nutrition. In countries that do not use antibiotics in beekeeping, bee gut bacteria contained far fewer resistance genes. The tetracycline resistance that we observed in American samples reflects the capture of mobile resistance genes closely related to those known from human pathogens and agricultural sites. Thus, long-term treatment to control a specific pathogen resulted in the accumulation of a stockpile of resistance capabilities in the microbiota of a healthy gut. This stockpile can, in turn, provide a source of resistance genes for pathogens themselves. The use of novel antibiotics in beekeeping may disrupt bee health, adding to the threats faced by these pollinators.