We characterized antimicrobial susceptibility profile and common virulence genes of S.suis isolated from blood or CSF samples of patients in Cambodia, Laos and Southern Viet Nam. A total of 13 patients from Cambodia, 13 from Laos and 52 from Southern Vietnam were identified with the mean age of 58.8 years (67.0% female), 46.3 years (23.3% female) and 48.6 years (20.0% female), respectively. All isolates were serotype 2. Isolates from Cambodia and Vietnam belonged to ST1 while 46% of Laos isolates belonged to ST104. Resistance to at least three drug classes macrolides, clindamycin and tetracyclin was detected in 30.8%, 87.5% and 92.9% of isolates from these three countries, in previous order. Tetracyclin resistance was correlated with tetM (100.0%, 12.5%, 94,7%) and tetO (16.0%, 87.5%, 22.8%) in Cambodia, Laos and Viet Nam isolates, respectively while TetL was only found in 7.0% of Viet Nam isolates. The macrolide-resistant gene ermB was present in 100.0%, 10.5% and 25.0% in isolates from Cambodia, Laos and Vietnam. Only genes sly was found in ST104 isolates while epf were found in 57.9%, 77.0% and 98.0% in Cambodia, Laos and Vietnam. S.suis caused meningitis and sepsis in human in Cambodia, Laos and Viet Nam, were associated with complex multi-antibiotic resistance, but still susceptible to ceftrizone and penicillin, two currently used antibiotics for human S. suis infection treatment.