This study was conducted to clarify the antimicrobial susceptibility and antibiotic resistance genes of Escherichia coli O8 and O9 isolated from 244 cattle feces collected from December 2021 to March 2022 in the Mekong Delta, Vietnam. E. coli was isolated from the feces following the method of TCVN 7686:2007 (ISO16654:2001), and serotypes O8 and O9 were identified by the PCR method. E. coli O8 and O9 were identified at a relatively high rate, with 15.98% and 8.20%, respectively. The disc diffusion method was applied to determine the antimicrobial susceptibility of those E. coli strains against thirteen antibiotics following the guidelines of the Clinical and Laboratory Standard Institute. Those E. coli O8 and O9 strains showed high susceptibility to amikacin (98.31%), doxycycline (96.61%), ofloxacin (94.92%), and levofloxacin (93.22%). However, those isolates exhibited resistance against ampicillin (47.46%), streptomycin (44.07%), and tetracycline (42.37%). Of those 59 E. coli strains, 35 strains (59.32%) were multiple resistant against two to twelve antibiotics with 25 resistant patterns. The pattern of Am+Ac+Co+Sm+Te+Cl was the most common type (6.78%). The prevalence of six antibiotic-resistance genes was determined by using PCR. Gene tetA was the most prevalent gene (66.10%), while cat1 was the least one (5.08%). Forty-five E. coli O8 and O9 strains (76.27%) harbored from two to four antibiotic resistance genes, and the pattern of strA+tetA+sulII was detected at the highest rate (23.73%). Therefore, the prevalence and antibiotic resistance of E. coli O8 and O9 should be controlled to protect cattle and human health.