Capsinoids are low-pungent capsaicinoid analogues in chili pepper fruits. They exhibit various bioactivities in humans similar to capsaicinoids, but do not produce a nasty burning sensation, encouraging their application in foods and supplements. Previous reports demonstrated that loss-of-function of putative aminotransferase (pAMT) leads to low-pungency and capsinoid accumulation. Therefore, the pamt allele is a useful gene in chili pepper breeding programs to enhance health-promoting properties. Eight loss-of-function alleles have been identified in the Capsicum genus, but the variation in pamt alleles remains to be fully elucidated. In this study, we identified one novel loss-of-function allele from the analysis of low-pungent chili pepper ‘No. 4034’ (C. chinense). ‘No. 4034’ contained mainly capsinoid with an undetectable level of capsaicinoid. A genetic complementation test was conducted by crossing ‘No. 4034’ with other accessions. The results indicated that ‘No. 4034’ possessed a loss-of-function pamt allele. Sequence analysis showed that the novel mutant allele contained a 7-bp insertion (TCGGTAC) in the 16th exon region, which we designated as pamt9. The insertion caused a frameshift mutation and resulted in a truncated protein. Gene expression analysis showed that the expression level of pAMT specifically decreased among biosynthetic genes tested here in ‘No. 4034’, compared with that of pungent accession. pamt9 will be useful for low-pungency and capsinoid breeding, and will provide additional information for variations in pAMT mutants.