ABSTRACT: In order to clarify the molecular mechanisms of drought tolerance between different maize (Zea mays L.) varieties at the protein level, iTRAQ (the isobaric tags for relative and absolute quantitation) quantitative proteomics were used for the comparative analysis of protein expression in the seedling roots of the drought-tolerant Chang 7-2 and drought-sensitive TS141 maize varieties under 20% PEG 6000 (polyethylene glycol 6000)-simulated drought stress. A total of 7723 proteins were identified using iTRAQ in the maize seedling roots. We detected 39371 peptides and 30148 unique peptides. The identified protein molecular mass was mainly distributed between 10–60 kDa. COG analysis divided these proteins into 24 categories, among which general function prediction contained the largest number of proteins (19.1%), followed by posttranslational modification, protein turnover, and chaperones (10.52%). In the identified differentially expressed proteins, 1552 of which were significantly differentially expressed. Of these, 1249 were differentially expressed in Chang 7-2 following drought stress, 577 of which were up-regulated and 672 were down-regulated. Four hundred and twenty five differentially expressed proteins were identified in TS141, 249 of which were up-regulated and 176 were down-regulated. Of these proteins, 32 demonstrated opposite expression levels in the two varieties, and there were 56 up-regulated proteins that were common between the two varieties. Comparing the Chang 7-2 treatment group and control group, the DEPS in the cellular component category were mainly enriched in cytoplasmic membrane-bounded vesicles, cytoplasmic vesicles, membrane-bound vesicles and vesicles; in the molecular function category, the DEPs were mainly enriched in cation binding and metal ion binding; and in the biological process category, the major enriched categories included phosphate-containing compound metabolic process, single-organism transport cellular component organization or biogenesis, carbohydrate metabolic process, and cellular component organization. Comparing the TS141 treatment group and control group, the DEPS in the cellular component category were primarily enriched in cytoplasmic membrane-bounded vesicles, cytoplasmic vesicles, membrane-bound vesicles, and vesicle; in the molecular function category, the DEPs were mainly enriched in cation binding and metal ion binding; and in the biological process category, the major enrichment categories included oxidation-reduction process and carbohydrate metabolic process. In Chang 7-2, the differentially expressed proteins were associated with ribosome pathway, glycolysis/gluconeogenesis pathway, and amino sugar and nucleotide sugar metabolism. In TS141, the differentially expressed proteins were associated with metabolic pathway, phenylpropanoid biosynthesis pathway, and starch and sucrose metabolism.