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As in other countries, in the Czech Republic, <i>Streptococcus suis</i> infection in pigs is considered an economically significant disease for the pig industry, though little is known about its population structure. We collected <i>S. suis</i> isolates from 144 farms in the years 2018–2022. All samples were taken from animals suffering from symptoms indicating possible <i>S. suis</i> infection. Serotyping revealed the presence of 23 different serotypes, and 18.94% were non-typable strains. The most common was S7 (14.96%), while other serotypes had frequencies of less than 10%. Sequence typing identified 56 different sequence types, including 31 newly assigned sequence types together with 41 new alleles in genes in the MLST schema. A large portion of isolates (25.70%) were of unknown sequence type. The most common sequence types were ST29 (14.77%) and ST28 (10.04%); the other sequence types had frequencies of less than 10%. In total, 100 different combinations of serotypes and sequence types were identified. Among them, S7ST29 was found in 72 isolates, representing 13.63% of all isolates, and was significantly associated with the central nervous system. Many other isolates of particular serotype and sequence type combinations were found in a few cases, and a number of isolates were non-typable.