This study assessed the recrudescence of Enterococci in mastitis cow's milk and investigated their resistance to vancomycin. 300 samples were compiled from separate places and fields in Kirkuk, Iraq during the period from February to May, 2022. The samples were ioculated on the surface of bile esculin agar plates with sodium azide and then incubated at 37°C for 24–48 h. The characteristic pin-point colonies with a zone of black precipitate and morphologically resembling Enterococci spp. were further subjected to presumptive identification by Gram' staining, catalase, and oxidase tests. All isolates were kept in BHIB with 30% glycerol at −70°C for further molecular detection. Enterococci isolates were tested for their susceptibility to different antibiotics by a disc diffusion technique. Based on the results of the sensitivity test, the ten isolates with the highest level of multiple resistances were selected from each of E. faecalis and E. faecium to examine the vanA, vanB genes by cPCR.The results of the bacteriological examination revealed that, 61 isolates (20.3%) of Enterococci According to phenotypic criteria; 42isolates were E. faecalis and 19 were E. faecium. Add this to the confirmatory tests that revealed 25 isolates (8.3%) were E. faecalis and 10 isolates (3.3%) were E. faecium detected by PCR. Antimicrobial susceptibility tests indicated high levels of multi-resistant E. faecalis and E. faecium strains. Vancomycin-resistant strains were 40% and 30% for E. faecalis and E. faecium, respectively. The genetic sequences of E. faecalis and E. faecium isolates and phylogenetic trees were established and registered in GenBank-NCBI. They obtained accession numbers (OP566382) for E. faecium and (OP566380) for E. faecalis, which became references in Iraq and around the world.