Streptococcus suis is a pathogen that causes invasive infections in humans and pigs. In this study, 448 S. suis isolates recovered from human infections in Thailand were characterized with regard to their antimicrobial susceptibility and antimicrobial resistance genes, including, for non-penicillin-susceptible isolates, sequence analyses of five genes encoding penicillin-binding proteins (pbp1a, pbp1b, pbp2a, pbp2b, and pbp2x). All 448 isolates were susceptible to cefepime and ceftriaxone, whereas 99.6%, 91.7%, and 72.9% of the isolates were susceptible to levofloxacin, penicillin, and chloramphenicol, respectively. Almost all isolates were resistant to tetracycline (98.2%), clindamycin (94%), erythromycin (92.4%), and azithromycin (82.6%). Genes tet(O) and ermB were the predominant resistance genes detected among macrolide- and tetracycline-resistant isolates. A total of 37 out of 448 isolates (8.2%) showed intermediately resistance to penicillin. Most of these isolates (59.5%) belonged to serotype 2-ST233. Comparison of the predicted translated sequences of five PBP proteins of a penicillin-susceptible isolate (strain P1/7) to the respective PBP sequences of ten non-penicillin-susceptible isolates revealed multiple amino acid substitutions. Isolates of CC221/234 showed highly variable amino acid substitutions in all PBP proteins. An ST104 isolate had a higher number of amino acid substitutions in PBP2X. Isolates belonging to CC233/379 had numerous substitutions in PBP2B and PBP2X. ST25 isolates exhibited fewer amino acid substitutions than isolates of other STs in all five PBPs. The antimicrobial resistance of S. suis is increasing worldwide; therefore, restrictions on antimicrobial use, continuous control, and the surveillance of this bacterium throughout the pork supply chain are crucial for ensuring public health and must be a priority concern.