Nucleases catalyze the cleavage of phosphodiester bonds in nucleic acids. TatD DNase is a conserved evolutionary protein expressed in various organisms, but its function has not been completely recognized. The aim of this research was to identify tatD genes in Bacillus sp. B2 isolated previously from soil in Damghan. The molecular identification of the isolate was performed by 16S rRNA amplification and sequencing. Two sets of degenerate primers were designed for tatD genes, and the amplified sequences were analyzed with BLAST. Deduced amino acid sequences of the tatD genes were aligned with the known TatD DNase from E. coli. The secondary structure of the TatD proteins and the presence of conserved domain were analyzed through PSIpred and CD-Search tools, respectively. The 16S rRNA sequence of Bacillus sp. B2 revealed 100% identity to the sequences of B. cereus, B. thuringiensis, and B. anthracis. Moreover, the amplified tatD1 and tatD2 genes showed more than 90% identity to tatD DNase of Bacillus species. The alignment of the obtained TatD sequences with TatD DNase from E. coli showed that metal ion-binding and catalytic residues were conserved among them. Secondary structures of the TatD1 and TatD2 were similar to the TatD DNase of E. coli, and CD-Search indicated that both TatD proteins belonged to metallo-dependent hydrolases superfamily. According to these results, the Bacillus sp. B2 isolate had two different tatD genes. It seems that tatD has been duplicated and evolved in the genome of some Bacillus species. Identification and characterization of TatD from various organisms is valuable for understanding its role as a conserved evolutionary protein.