Local Thai and imported Malaysian beef in southern Thailand area carry several Shiga toxin-producing Escherichia coli (STEC) serotypes. STEC O104 is an important pathogen capable of causing outbreaks with considerable morbidity and mortality. This study investigated the presence of E. coli O104 from local Thai and imported Malaysian beef obtained from markets in Hat Yai City, Songkhla Province during August 2015 - February 2016. Thirty-one E. coli O104 strains were isolated from 12 beef samples (16% and 23% Thai and imported Malaysian, respectively). Thirty strains possessed aggA (coding for a major component of AAF/I fimbriae), a gene associated with enteroaggregative E. coli (EAEC) pathotype, and all strains carried fimH (encoding Type 1 fimbriae). Thirty strains belonged to phylogenetic group B1 and one strain (from Malaysian beef) to group A. Agglutination of yeast cells was observed among 29 E. coli O104 strains. Investigation of stx2 phage occupancy loci demonstrated that sbcB was occupied in 12 strains. Antimicrobial susceptibility assay revealed that 7 strains were resistant to at least one antimicrobial agent and two were multi-drug resistant. One strain carried extended spectrum β-lactamase gene blaCTX-M and three carried blaTEM. PFGE-generated DNA profiling showed identical DNA pattern between that of one EAEC O104 strain from Thai beef and another from Malaysian beef, indicating that these two strains originated from the same clone. This is the first report in Thailand describing the presence of EAEC O104 from both Thai and imported Malaysian beef and their transfer between both countries. Thorough surveillance of this pathogen in fresh meats and vegetables should help to prevent any possible outbreak of E. coli O104.