Clostridium tyrobutyricum is a rod-shape, gram-positive bacteria that grows under anaerobic condition and produces butyric acid, acetic acid and hydrogen as their major products. Two mutants (PPTA and PACK) were created by integrational recombinant, and the third mutant HydEm was created by adaptation. All of these mutants have improved butyric acid and/or Hydrogen production. Shotgun microarray technology was employed to investigate the gene expression pattern among these strains. A genomic library was constructed, the insert were amplified by PCR, and then printed onto poly-lysine coated glass slide. Cy3 and Cy5 labeled cDNA were used for competitive hybridization. After the transcriptional analysis, the genes fall into several clusters of co-regulated genes by the difference expression pattern among the strains. The information will be used for further research.