In this study, we used DNA sequencing to study species identification and phylogeny of German cockroach that has medical importance, So, the comparisons were made with specific region of mitochondrial ribosomal RNA gene (16S rRNA gene) of two collected Blattella germanica the first from central city of Mashhad (GM), Iran, and the second from city of Tehran (GT), Iran, and other individuals of B. germanica from different areas, that already have been recorded in the BLAST database, on the basis of the Single Nucleotide Polymorphisms (SNPs), nucleotide substitutions, and DNA and putative amino acid sequence data. Also, a phylogeny for these cockroaches based on the DNA sequence of the 16S rRNA genes is offered. Our results indicated that the nucleotide sequence of the B. germanica (GM) matched with sequences from other subspecies, except the (thymine–adenine) SNPs at position 15 and 19 of the 376-bp, and the nucleotide sequence of B. germanica (GT), also, matched with sequences from other subspecies, except (adenine-guanine) SNP at position 360-bp, and (thymine-guanine) SNP at position 368-bp of a portion of the 16s rRNA gene. Therefore, the B. germanica (GM), B. germanica (GT), and B. germanica isolates in BLAST database were different among them. Furthermore, we found that the forward (LR-J-13017) primer and reverse (LR-N-13398) primer were very impressive to distinguish among cockroaches belong sub-order Dictyoptera especially German cockroach. As well, these primers were produced a small DNA fragment of the 16S rRNA gene that includes all SNPs required for B. germanica isolates identification.

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