In this study, F₁ population was constructed by a cross derived from tobacco cultivars G117 and RG13. A natural mutant individual was found in F₃ lines during breeding selection. The mutant exhibits a yellow-green leaf phenotype, which is controlled by one recessive gene by classical genetic analysis. Significant markers related with aimed trait were detected by jointly 430 K SNP chip with two extreme selective populations. Subsequently, the entire segregation population derived from one F₇ plant was genotyped using PCR method at these significant markers loci. Finally, the recessive gene controlled a yellow-green leaf phenotype in tobacco was mapped to chromosome 5 and co-segregated with the SNP marker M7. These results could enhance our understanding of the inheritance of yellow-green leaf mutant, and provide a new avenue for mapping the mutant loci or the QTL in tobacco by combining SNP chip and extreme selection populations.