Using newly developed methods and software, association mapping was conducted for chromium content and total sugar in tobacco leaf, based on four-omics datasets. Our objective was to collect data on genotype and phenotype for 60 leaf samples at four developmental stages, from three plant architectural positions and for three cultivars that were grown in two locations. Association mapping was conducted to detect genetic variants at quantitative trait SNP(QTS) loci, quantitative trait transcript(QTT) differences,quantitative trait protein(QTP) variability, and quantitative trait metabolite(QTM) changes,which can be summarized as QTX locus variation. The total heritabilities of the four-omics loci for both traits tested were 23.60% for epistasis and 15.26% for treatment interaction.Epistasis and environment × treatment interaction had important impacts on complex traits at all-omics levels. For decreasing chromium content and increasing total sugar in tobacco leaf, six methylated loci can be directly used for marker-assisted selection, and expression of ten QTTs, seven QTPs and six QTMs can be modified by selection or cultivation. Using newly developed methods and software, association mapping was conducted for chromium content and total sugar in tobacco leaf, based on four-omics datasets. Our objective was to collect data on genotype and phenotype for 60 leaf samples at four developmental stages, from three plant cultivation positions and for three cultivars that were grown in two locations. Association mapping was conducted to detect genetic variants at quantitative trait SNP (QTS) loci, quantitative trait transcript (QTT) differences, quantitative trait protein (QTP) variability, and quantitative trait metabolite (QTM) changes, which can be summarized as QTX locus variation. The total heritabilities of the four-omics loci for both traits tested were 23.60% for epistasis and 15.26% for treatment interaction. Epistasis and environment × treatment interaction had important significant on complex traits at all-omics levels. For decreasing chromium content and increasing total sugar in tobacco leaf, six methylated loci ca n be directly used for marker-assisted selection, and expression of ten QTTs, seven QTPs and six QTMs can be modified by selection or cultivation.