I would suggest a combination of 16S rRNA gene sequencing along with 16S-23S ITS for proper species discrimination for Bacillus.

For details please refer to: Xu and Cote (2003) Phylogenetic relationships between Bacillus species and related genera inferred from comparison of 3′ end 16S rDNA and 5′ end 16S–23S ITS nucleotide sequences. IJSEM 53 (3) 695-704.

16S RNA sequencing method can be employed to distinguish the bacteria at species level

Yes, I did 16S rDNA sequence. After the sequence you only know the genera. But how to the strain belonging to that genera, because i have many isolates

I would suggest a combination of 16S rRNA gene sequencing along with 16S-23S ITS for proper species discrimination for Bacillus.

For details please refer to: Xu and Cote (2003) Phylogenetic relationships between Bacillus species and related genera inferred from comparison of 3′ end 16S rDNA and 5′ end 16S–23S ITS nucleotide sequences. IJSEM 53 (3) 695-704.

16 S RNA sequencing method can be usefull and more detailed genotyping, which specific for your bacteria

Yes I agree with the sanjay antony's answer.....u can use combination of 16S rRNA and 16s-23s ITS....

Yes. You should follow the Multilocous sequence typing using different houskeeping genes.

16S rRNA gene sequencing with multiple internal primers would be the best way to describe bacterial isolates in species level...

you can try RFLP method to confirmation to the same genus level and if you want to know upto species level you have to go for 16s rRNA gene sequencing............

a combination of 16S rRNA gene sequencing along with 16S-23S ITS for proper species discrimination for Bacillus will be good.

You can use inter-tRNA and BOX-PCR or singe-adapter AFLP to group the strains on species and sub-species level, pick up representative strains for each group and make MLST for at least 4 genes. There are some species in Bacillus that differe less than 1% in 16S rRNA and 5% in IST region.

You can consider biochemical assay for species differentiation. Modernized form of biochemical assays such as Biolog GEN III Microplate contains 94 biochemical tests in a single 96-well plate to profile and identify a broad range of Gram-negative and Gram-positive bacteria including the Bacillus genera. It can distinguish over 100 species belonging to the Bacillus genera. The only downside is the microplate needs to be analyzed by a specialized microlpate reader (also from Biolog) and compared against a database using Biolog’s Microbial Identification Systems Software for specie identification (quite a bit of an investment here). However, if your local distributorship is willing enough, you might just be able to get their help with the identification part.

You can find more information here: http://www.biolog.com/products/?product=Microbial%20ID%20%2F%20Characterization&view=Product%20Literature