This study was included molecular study of Hyalomma anatolicum (Ixodoidea: Ixodidae) based on 16S rRNA gene collected from three regions' in Babylon province. The results of gel electrophoresis image (1.5 % agarose) shows the amplicons of a partial region within large subunit RNA gene (size= 450 bp). The sequencing data has reported species of H. anatolicum 16S rRNA gene (OQ162293) to OQ162298 these species revealed close matching on the phylogenetic tree to an isolate of H. anatolicum for different countries. This study represents first in Babylon Governorate to diagnose H. anatolicum by targeting 16S rRNA gene.