Non-O157 Enterohaemorrhagic Escherichia coli (EHEC) has become a major public health concern all around the globe. This study investigated the E. coli serogroup O45 from raw meats in southern Thailand using the immunomagnetic separation approach (IMS). Forty-nine E. coli O45 strains were obtained from 13 positive meat samples (13/105), showing the prevalence of O45 contamination as 12.4%. They were not members of EHEC. Exploration of other virulence genes exhibited that fimH, responsible for bacterial adherence, was found in all strains, while astA encoding for EAST-1 toxin was 63.2% of the strains. Moreover, lpf encoding long polar fimbriae was found as 30.6%. Phylogenetic group analysis revealed that the majority of E. coli O45 belonged to group D (88%), followed by group A (8%) and group B1 (4%). However, none belonged to group B2. The intactness of stx2-phage integration sites showed that sbcB was occupied by prophages at the highest rate, followed by the Z2577 site. Antimicrobial susceptibility assay demonstrated relatively high bacterial resistance to cephalothin (78%), streptomycin (51%), cotrimoxazole (39%), tetracycline (31%), and chloramphenicol (23%). Furthermore, multi-drug resistant ability was uncovered in O45 strains at 49%. DNA profiling of E. coli O45 by BOX-PCR analyzed at 80% genetic similarity revealed 5 distinguishable clusters. More importantly, the strains from different samples and time intervals showed identical DNA fingerprints, suggesting that they may have originated from the same bacterial clone.