The diploid potato species Solanum chacoense has been used for over three decades to introgress leptine glycoalkaloid mediated Colorado potato beetle (Leptinotarsa decemlineata) host plant resistance into cultivated potato (Solanum tuberosum). Investigation and exploitation of the complex genetic basis conferring Colorado potato beetle resistance in S. chacoense has been obscured by interspecific crosses, low recombination within small populations, and a dearth of marker density to provide sufficient resolution. As a consequence, no cultivars resistant to this devastating insect defoliator pest have been achieved. To combat these challenges, we generated a self-compatible, diploid F2 mapping population of 236 individuals from the S. chacoense lines USDA8330-1 and M6, a self-compatibility donor, segregating for Colorado potato beetle field resistance and accumulation of secondary metabolites implicated in Colorado potato beetle resistance. We employed bi-parental linkage mapping and whole genome bulk segregant analysis in this F2 population to identify a major QTL on the long arm of chromosome 2 associated with Colorado potato beetle field resistance.