We recently described a new source of host-plant resistance to the Colorado potato beetle, Leptinotarsa decemlineata (Say), in a tetraploid potato (Solanum tuberosum L.) selection, ND2858–1. This genotype, and selected backcross progeny, had little damage while check cultivars were defoliated in open-choice field assays. To further characterize the observed deterrence, we determined foliar glycoalkaloids and conducted no-choice assays with ND2858–1 backcross progeny genotypes (ND4382-n). Development of neonate L. decemlineata in detached leaf assays on resistant progeny genotypes was delayed and larval weight gain after 4 d was inhibited by 75% relative to larval development and weight gain on susceptible genotypes. Inhibition of larval development in detached leaf assays with the selected progeny genotypes was equivalent to that of high-leptine genotypes of S. chacoense Bitter. Foliar glycoalkaloids of resistant genotypes included low levels of leptines I and II. The unlikely nature of this cross and the presence of leptine in this and resistant progeny selections cast doubt on the recorded pedigree. Molecular analyses were conducted by restriction fragment-length polymorphism and amplified fragment-length polymorphisms. Both methods established a high degree of relatedness to S. tuberosum and S. chacoense but not to S. fendleri. We conclude that ND2858–1 did not originate from a cross with S. fendleri, but is likely derived from S. chacoense. Oviposition and larval survival were reduced when adult L. decemlineata were placed in cages with resistant genotypes; an effect that was enhanced by inclusion of Perillus bioculatus F. Therefore, the nonpreference previously observed in open-choice field defoliation assays is also associated with antibiotic effects on L. decemlineata. The resistance may be caused by leptines, but is greater than would be expected by the leptine content. This source of host plant resistance could be a cost-effective management strategy, especially if combined with other resistance mechanisms or compatible control measures to delay development of resistance in the target insects.