Members of the bacterial order Bacillales have been of great interest for agricultural, horticultural, industrial and medical applications because of their capacity to produce various extracellular enzymes. One of the challenges for Bacillales study is to rapidly and effectively identify and characterize newly isolated strains. In the present study, Raman spectroscopy was performed to identify 14 Bacillales strains isolated from Tibet, China. The biochemical properties of each isolate were characterized, and several Raman bands corresponding to nucleic acids, proteins or saccharides were different between isolates. Multivariate analysis of 112 Raman spectra clearly revealed that all 14 isolates were clustered into 3 groups, which was in accordance with the phylogenetic analysis of their 16S rRNA genes. Our results suggest that Raman spectroscopy is an effective and promising approach that could quickly discriminate different phylogenetic groups of Bacillales.