High content of leptine glycoalkaloids present in Solanum chacoense has been associated with genetic resistance to Colorado potato beetle (Leptinotarsa decemlineata [Say]). From an unrecorded accession of S. chacoense, the North Dakota State University breeding program has developed a tetraploid genotype, ND4382-19, that contains foliar leptines. In this study, using a segregating population, ND5873 (ND4382-19 x Chipeta), and GC-MS to analyze foliar content of alkaloids, two loci, involved in the synthesis of leptines were identified. They segregated as two complementary epistatic genes that allowed the synthesis of leptinidine (Lep) and acetyl-leptinidine (AL), respectively. Partial AFLP maps for both parents were developed using 97 individuals from population ND5873. The total lengths mapped for ND4382-19 and Chipeta were 1,883 and 1,021 cM, respectively. The marker for Lep was located at the distal end of simplex-coupling linkage group R37. Expansion of the initial mapping population and analysis of Lep-containing individuals allowed us to identify the linkage group (R35) that enabled synthesis of AL. By the use of simple sequence repeat markers, linkage group R37 (Lep) and linkage group R35 (AL) have been identified as homologs of chromosomes II and VIII, respectively.