Streptococcus pasteurianus is an opportunistic pathogen affecting various animals and is an underreported zoonotic threat. It is also a causative agent of swine streptococcosis and can be co-detected with Streptococcus suis, another significant pig and zoonotic pathogen. However, the dynamics of co-colonization between these pathogens, along with the genomic features and antibiotic resistance profiles of S. pasteurianus, remain poorly understood. In this study, we developed a multiplex PCR (mPCR) assay to detect S. pasteurianus and S. suis in 827 tonsil samples from healthy pigs, with 81 samples positive for both pathogens. Pan-genome analysis revealed an open pan-genome, indicating an adaptable genome. Antibiotic resistance gene analysis identified 21 distinct genes, including the first identification of mef(A), msr(D), optrA, lnu(G), spw, dfrF, and fexA in S. pasteurianus. Strain WUSP082 carried 15 resistance genes, many of which were located on mobile genetic elements. ICE