Abstarct: Entomopathogenic fungi are widely distributed throughout the fungal kingdom. Some insect-pathogenic fungi have restricted host ranges, while others have a wide host range. Several species of fungi are potent biocontrol agents of plant pathogenic fungi and arthropods. mextarhizium spp.an entomopathogenic fungus well characterized for the biocontrol of a wide range of pest. mextarhizium spp. mainly identificated baxsed on the morphological and molecular characteristics. The objectives of this work were to isolate and identify varieties with ITS region. We investigated 22 isolates which collected from different soil from different sites of Shahrood and North of Iran. These isolates have been partially characterized using morphological traits such as Growth rate, Sporulation, pathogenicity against Tribolium castaneum (Col, Tenebrionidae). Morphological experiments indicated that there are correlation between 3 parameters. Internal transcribed spacer (ITS) and 5.8S rDNA sequences analysis was employed to identify species and genotypes. The ITS regions were amplified using the ITS1 and ITS4 primers that was a unique fragment of approximately 540 bp. The sequences were aligned using the Codon Code Aligner program and together with 12 isolates of mextarhizium available in the GenBank used for draw tree in Mega (Maximum Parsimony with 1000 bootstrap). Among 8 Clusters, 5 Clusters have 25 isolates of Shahrood and environs, didn’t have any match known species and there are possibility of quality of 5 new species in these areas that need more molecular study with another markers.5 identified isolates belong to M. flavoviride, M. anisopliae, M. robertsii, M. guizhouense and M. velutinum.