The discovery of antibiotics more than 70 years ago initiated a period of drug innovation and implementation in human and animal health and agriculture. These discoveries were tempered in all cases by the emergence of resistant microbes1,2. This history has been interpreted to mean that antibiotic resistance in pathogenic bacteria is a modern phenomenon; this view is reinforced by the fact that collections of microbes that predate the antibiotic era are highly susceptible to antibiotics3. Here we report targeted metagenomic analyses of rigorously authenticated ancient DNA from 30,000-year-old Beringian permafrost sediments and the identification of a highly diverse collection of genes encoding resistance to β-lactam, tetracycline and glycopeptide antibiotics. Structure and function studies on the complete vancomycin resistance element VanA confirmed its similarity to modern variants. These results show conclusively that antibiotic resistance is a natural phenomenon that predates the modern selective pressure of clinical antibiotic use.