Salmonella enterica serovar Typhi, a gram-negative human enteroinvasive pathogen is the etiological agent of typhoid fever, and is an important experimental model for prokaryote research. DNA microarray technology was widely used in analysis of genomic structures and expression profiles. The genomic DNA microarrays were prepared based on the genomic sequences of S. enterica serovar Typhi. A total number of 4 201 protein encoding genes selected from strains Ty2, LT18 and a z66⁺ wild strain of S. enterica serovar Typhi were amplified by PCR. The products were purified and printed onto the poly-L-lysine coated chip slides in duplicate to form the genomic DNA microarrays. Fluorescently labeled probes were prepared by priming of genomic DNAs with random hexamers and extension with Klenow DNA polymerase. Labeled DNAs were hybridized with the microarrays to check the printing effect and verify the genes order. The genomic DNA microarray-based bacterial gene expression profiling analysis was optimized and used to investigate global transcriptional responses when wild strain of S. enterica serovar Typhi was grown in high- and low-osmotic environmental conditions. The major results were consistent with those of previous research with oligo-genomic microarrays of S. enterica serovar Typhi. These results demonstrated that S. enterica serovar Typhi genomic DNA microarrays were successfully prepared and could be utilized in relative gene expression profiling analysis and comparative genomic researches.