Transgenic plants producing environmentally benign Bacillus thuringiensis (Bt) toxins are deployed increasingly for insect control, but their efficacy will be short-lived if pests adapt quickly. The diamondback moth (Plutella xylostella), a worldwide pest of vegetables, is the first insect to evolve resistance to Bt toxins in open-field populations. A recessive autosomal gene confers resistance to at least four Bt toxins and enables survival without adverse effects on transgenic plants. Allelic variants of this gene confer resistance in strains from Hawaii, Pennsylvania, and the Philippines. Here we exploited the biphasic nature of Lepidopteran genetic linkage to map this gene in diamondback moth with 207 amplified fragment length polymorphisms as DNA markers. We also cloned and sequenced an amplified fragment length polymorphism marker for the chromosome containing the Bt resistance gene. The results provide a powerful tool for facilitating progress in understanding, monitoring, and managing resistance to Bt.