Leptinotarsa decemlineata, the Colorado potato beetle (CPB), is a herbivore that primarily feeds on Solanum foliage and is a global pest of the potato agricultural industry. Potato breeding through cross-hybridization with CPB-resistant wild relatives is used for genetic improvement. The wild species Solanum okadae was demonstrated to deter CPB feeding in choice and no choice feeding assays. Liquid chromatography–mass spectrometry (LC-MS) was used for comparative metabolite profiling between S. okadae and CPB-susceptible domesticated potato variety, Solanum tuberosum cv. Shepody. Major foliar metabolites detected were steroidal glycoalkaloids (SGAs) with tomatine and dehydrotomatine produced in S. okadae and solanine and chaconine in S. tuberosum cv. Shepody. Cardiac glycosides were also detected in the foliar metabolite profile of S. okadae but not S. tuberosum cv. Shepody. This class of plant compounds have known insecticidal activity through inhibition of animal Na+/K+ ATPase. Thin-layer chromatography (TLC) separation of foliar extracts also provided evidence for cardiac glycosides in S. okadae. Cardiac glycosides are known inhibitors of Na+/K+ ATPase, and foliar extracts from S. okadae (OKA15), but not S. tuberosum cv. Shepody, were able to inhibit the Na+/K+ ATPase of CPB. These findings suggest a novel mechanism of plant resistance against CPB involving production of cardiac glycosides in S. okadae.