Identification of a novel pungency-controlling gene Pun3, which acts as a master regulator of capsaicinoid biosynthetic genes in Capsicum annuum. Capsaicinoid is a unique compound that gives hot peppers (Capsicum spp.) their spicy taste. The Pun1 and Pun2 loci are known to control pungency in Capsicum species. Whereas Pun1 encodes an acyltransferase, the identity of Pun2 is currently unknown. Here, we used recombinant inbred lines and F2 plants derived from a cross between the non-pungent C. annuum accession 'YCM334' and the pungent C. annuum cultivar 'Tean' to identify a novel non-pungency locus. Inheritance studies showed that non-pungency in C. annuum 'YCM334' is controlled by a single recessive gene, which we named Pun3. Using a high-density SNP map derived from genotyping-by-sequencing, Pun3 was mapped to chromosome 7. By comparing physical information about the Pun3 region in the C. annuum 'Zunla-1' and C. chinense 'PI159236' reference genomes, we identified candidate genes in this target region. One cDNA sequence from 'PI159236' was homologous to an unannotated gene in 'Zunla-1.' This sequence was also homologous to CaMYB31, which is expressed only in 'Tean' and harbors one stop codon in the non-pungent accession 'YCM334.' RNA-Seq analysis showed that major structural genes in the capsaicinoid biosynthetic pathway were significantly downregulated in 'YCM334' compared to pungent pepper. Therefore, CaMYB31 is a candidate gene for Pun3, which may act as a master regulator of capsaicinoid biosynthetic genes in pepper.