Several genes regulating capsaicinoid biosynthesis including Pun1 (also known as CS), Pun3, pAMT, and CaKR1 have been studied. However, the gene encoded by Pun2 in the non-pungent Capsicum chacoense is unknown. This study aimed to identify the Pun2 gene by genetic mapping using interspecific (C. chacoense × Capsicum annuum) and intraspecific (C. chacoense × C. chacoense) populations. QTL mapping using the interspecific F2 population revealed two major QTLs on chromosomes 3 and 9. Two bin markers within the QTL regions on two chromosomes were highly correlated with the capsaicinoid content in the interspecific population. The major QTL, Pun2_PJ_Gibbs_3.11 on chromosome 3, contained the pAMT gene, indicating that the non-pungency of C. chacoense may be attributed to a mutation in the pAMT gene. Sequence analysis revealed a 7 bp nucleotide insertion in the 8th exon of pAMT of the non-pungent C. chacoense. This mutation resulted in the generation of an early stop codon, resulting in a truncated mutant lacking the PLP binding site, which is critical for pAMT enzymatic activity. This insertion co-segregated with the pungency phenotype in the intraspecific F2 population. We named this novel pAMT allele pamt11. Taken together, these data indicate that the non-pungency of C. chacoense is due to the non-functional pAMT allele, and Pun2 encodes the pAMT gene.