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The maize NAC gene, encoding a NAC domain transcription factor, plays vital roles in the growth process of maize under abiotic stresses. To investigate the response of ZmNAC33 to salinity stress, we analyzed the expression of the ZmNAC33 gene in roots and leaves of three-leaf stage seedlings among maize inbred lines. The result showed that the expression of ZmNAC33 was up-regulated in the salinity tolerant inbred line Ji853 and down-regulated in the salinity susceptible inbred line Chang7-2. The cDNA and the full sequence from maize inbred line Ji853 were amplified to analyze genetic structure. ZmNAC33 gene comprises three exons and two introns. We compared genetic diversity of ZmNAC33 among 69 elite maize inbred lines, a total of 144 sequence variants, including 113 SNPs and 31 Indels. Neutrality tests were negative selection by Tajima’s D and Fu and Li’s test. Analysis of polymorphism sites showed that at least 28 recombination’s have occurred. This finding provides insight into the development of breeding programs, germplasm management and association genetic studies.