BACKGROUND AND ABJECTIVE Mastitis is an inflammation of the mammary gland tissue because of various agents, the most important cause of the mastitis is the entry of pathogenic microorganisms, especially bacteria into the breast tissue and then reproduction and production of toxins by them. This disease can cause changes in the quality and quantity of milk, and for this reason, it is considered as one of the most important diseases in dairy cow’s industries. One of the most common pathogens associated with clinical mastitis in cattle is Mammary Pathogenic Escherichia coli (MPEC). The MPEC strains could be responsible for economic loss in the dairy cattle and has zoonotic importance as well. Several methods including serotyping, pathotyping and phylothping have been used for E. coli typing and assigning. The aim of the present study was to assignment of the phylogenetic group in MPEC isolated from milk sample of cows with clinical mastitis. MATERIALS AND METHODS The present study was carried out on MPEC strains (n= 65) isolated from milk sample of cows with clinical mastitis in industrial dairies of Hamadan county. First, genomic DNA was extracted from bacterial strains using rapid boiling method. Extracted DNA was then assigned to phylogenetic typing using the revised Clermont E. coli phylotyping method, a Quadruplex PCR based procedure. RESULTS AND DISCUSSION The Clermont E. coli phylotyping method results revealed that the 65% of MPEC isolates can be assigned to a phylogroups (A, B1, B2, C, D, E, F and Escherichia clade I), and 35% of isolates were not ascribable to any group. Out of 65 MPEC strains the predominant phylogroup was phylogroup B1 (48 %) followed by E (11 %). CONCLUSION Current study findings indicate that most predominant phylogroup of MPEC isolated from milk sample of cows with clinical mastitis in industrial dairies of Hamedan county was B1 phylogroup. Additionally, Clermont E. coli phylotyping method recommended as a useful and inexpensive genetic