Some lactic acid bacteria produce high molecular weight polysaccharides (EPS) during milk fermentation. It is unclear if there is any change in the properties of EPS during the fermentation. The molar mass of EPS was investigated during fermentation of milk with Streptococcus thermophilus (St-143). We used crude extracts, instead of highly purified samples, to observe more native EPS systems. Reconstituted nonfat milk samples were fermented by S. thermophilus at 40°C until pH 4.5. Crude extracts of EPS were obtained at 4 different fermentation times starting from 150 min (pH ~5.3) to 330 min (pH~4.5). Whey from fermented milk was ultrafiltered using 100 kDa membrane to remove soluble milk sugars and proteins. The UF retentate was centrifuged and supernatant was further concentrated using single pass pressure UF cell with 100 kDa membrane. The concentrated EPS solution was analyzed for protein and sugar contents. Molar mass of EPS was estimated using size exclusion chromatography-multi angle laser light scattering (SEC-MALLS). Polysaccharide content of the EPS concentrates varied from ~0.3 to 0.4 mg/mL. Protein impurities in the EPS concentrate were high when sampled at pH 5.3, and significantly decreased (P < 0.05) until pH 4.5. Extracts separated using SEC gave an initial peak that appeared to be mostly high M_w protein aggregates and a second peak that corresponded to EPS. The molar mass of EPS ranged from 1.57 × 10⁶ to 2.86 × 10⁶ g/mol. EPS sampled at pH 5.3 had significantly higher (P < 0.05) molar mass than samples obtained at pH 4.5. EPS samples also appeared to be monodisperse (i.e., M_w/M_n = 1). The radius of gyration of EPS was ~54 nm and was not significantly different between the different pH samples. It appears that the properties of EPS may vary during milk fermentation.