A change of layer arrangement of shoot apical meristem (SAM) organized by three cell layers (L1, L2 and L3) is thought to be one of the provocations of bud sport, which often induces changes in phenotypic colors in periclinal chimeras. This paper describes a cell layer rearrangement which is the cause of spontaneous flower color mutation by using two carnation (Dianthus caryophyllus L.) cultivars that are presumably periclinal chimeras, ‘Feminine Minami’ (deep pink flower) and its recessive sport ‘Tommy Minami’ (pinkish red flower). The genotype of the acyl-glucose-dependent anthocyanin 5-glucosyltransferase (AA5GT) which is responsible for the color change of red to pink, in each cell layer was deduced by genomic analysis using tissues originated from specific cell layer and investigation of partial petal color mutations. In the results, the genotype of the L1 of ‘Feminine Minami’ was heterozygous for functional AA5GT and non-functional AA5GT carrying retrotransposon Ty1dic1 (AA5GT-Ty1dic1), and its inner cell layer hid red flower genotype, whereas AA5GT-Ty1dic1 of the L1 of ‘Tommy Minami’ became homogenic in absence of the insertion of a new Ty1dic1. Our outcomes concluded that the L1 of ‘Tommy Minami’ harboring the recessive AA5GT alleles are attributed to the inner cell layer of ‘Feminine Minami’ possessing red flower genotype.