The pale yellow (PY) trait in tobacco (Nicotiana tabacum L.) was first described in 1969 and was found to be controlled by a single dominant gene. Plants containing a PY gene show accelerated chlorophyll breakdown compared to wild type plants. In commercial tobacco varieties, the accelerated chlorophyll breakdown trait mediated by the PY locus could help growers to better manage the crop and minimize losses due to unfavorable weather conditions and diseases. However, the PY locus and its associated PY gene are still uncharacterized, so that the inclusion of the PY locus in a breeding program results in a time-consuming and subjective selection process. In a previous study, we mapped the PY locus on the tobacco genome and identified single nucleotide polymorphism (SNP) markers to enable accelerated breeding of the trait. We generated and analyzed a mapping population (F₂ generation resulting from a cross between dark tobacco variety Narrow Leaf Madole LC [non-PY-variety] and TI1372 [source of the PY locus]) and found a quantitative trait locus (QTL) explaining 75 % of variance in the PY trait. Within this QTL region, putative candidate genes were identified by RNASeq differential gene expression analysis and confirmed with quantitative real time polymerase chain reaction (PCR). In the present study, we analyzed two candidate genes in more detail by knockdown (RNAi) and overexpression studies and could validate one gene as responsible for the phenotype. While overexpression lines showed delayed chlorophyll breakdown, RNAi lines had an accelerated chlorophyll breakdown phenotype with a lower leaf chlorophyll content and increased expression levels of leaf senescence-related genes. Our study demonstrates the utility of a high-density SNP array for mapping of traits and, most importantly, provides a key target gene to enable more precise and faster breeding of the PY trait in commercial tobacco cultivars.