Acyltransferases (Pun] gene) are biosynthetic systems involved in the synthesis of capsaicin produced by Capsicum. In order to develop molecular markers for rapid and accurate detection of capsaicin in peppers, we designed three primers (26F, 54F, and 1305R) targeted to specifically amplify Pun] gene from seedling tissues of hot peppers, sweet peppers, and hybrids (hot pepper X sweet pepper). Results showed that 26F/1305R primers and 54F/1305R primers amplified only a single PCR band of 1.7 kb and 1.0 kb from hot peppers and sweet peppers, respectively. PCR primers combinations 26F/1305R primers and 54F/1305R primers used in multiplex PCR detection in each progeny also amplified two PCR bands of 1.7 kb and 1.0 kb. The molecular characterization analyses proved to be accurate and sensitive for detection and breeding of capsaicin existence situation in pepper seedlings, without waiting for to fruit mature, tasting or HPLC analysis.