Leaf colour is an indicator of chlorophyll (Chl) level and isolating leaf colour mutants can facilitate the understanding of Chl metabolism regulation. Here we describe an ethyl methanesulfonate-induced light colour mutant white stem 1 (ws1) in common tobacco (Nicotiana tabacum L.) that shows a phenotype highly similar to Burley tobacco (Nicotiana tabacum L.), a type of air-cured tobacco which has light-coloured leaves with white veins. Compared with the wild type, the light green stem of ws1 gradually became pale white along with growth, while ws1 leaves lost green colour rapidly which was positively correlated with the decline of Chl levels. A series of genetic analyses indicated that the ws1 mutant phenotype was controlled by two recessive nuclear genes ws1a and ws1b which were preliminarily mapped to the intervals of tobacco simple sequence repeat markers linkage group 5 and 24 using the BC1F2 populations, respectively. The allelism test further revealed that the same two genes controlled the Burley character in Burley tobacco. Based on the Chl-deficient phenotype of ws1 and the locations of the two genes, we hypothesised that ws1a and ws1b were paralogs of each other probably originated from the ancestral species N. sylvestris and N. tomentosiformis, respectively. Both genes might share similar biological functions and expression patterns, and play key roles in the regulation of Chl biosynthesis. These results laid a solid foundation for marker-assisted selection breeding and gene function analysis of the Burley character in tobacco.