Hookworm infection remains one of the most important public health threats worldwide, with an estimated 800 million people infected. Heavy hookworm infection is the leading cause of anemia in the tropics, resulting in debilitating and sometimes fatal iron-deficiency anemia caused by blood loss to feeding adult worms in the intestine. Children, pregnant women, and the elderly are particularly susceptible to morbidity from hookworm infection. Control strategies are restricted to periodic de-worming of infected individuals with benzimidazole (BZ) anthelmintics. There is considerable concern that resistance to BZ drugs will develop with increased use in mass drug administration (MDA) programs to control hookworms. Molecular tests to monitor the emergence of resistance are necessary, but the genetic mutations that confer resistance to BZ are unknown in hookworms, and appear to be different than those that confer resistance in other parasitic nematodes. The lack of a BZ resistant strain of hookworm prevents identification of the resistance mutations. To determine the underlying genetic mutations that confer BZ resistance in hookworms, we propose using a novel in vitro mutagenesis and selection protocol to generate a strain of the canine hookworm Ancylostoma caninum that is phenotypically resistant to BZ. In Aim 1 we will mutagenize adult hookworms in vitro to generate genetic variation, and return them to a nave host to reproduce. F2 offspring will be selected for resistance by exposure to BZ drugs in vitro. Survivors will be used to infect new hosts, and the F3 offspring selected with BZ. F3 survivors will be reciprocally backcrossed to the wild type parental strain, and BZ used to select resistant worms for propagation of the resistant strains. Phenotypic resistance will be confirmed by standard egg hatch and larval development assays. In Aim 2, we will identify mutations associated with resistance by whole genome sequencing and comparison to the susceptible wild type parental strain. Candidate resistance alleles will be confirmed by their ability to confer BZ resistance on the model nematode Caenorhabditis elegans in trans. Generation of a resistant strain of hookworm and the identification of the genetic mutations that cause resistance to BZ anthelmintics will permit the development of molecular based tools to monitor the frequency of resistance alleles in populations undergoing treatment. This will allow modification of treatment regiments in time to prevent widespread resistance from developing.