► We study EPS from wild type and exoY strains of bacterium Sinorhizobium meliloti. ► EPS extracted by centrifugation, EDTA and formaldehyde/NaOH were compared. ► Two major components of proteinaceous and polysaccharide nature were found. ► Extraction methods influenced TOC, TN and total protein content in EPS. ► Molar mass distributions also differed among treatments and bacterial strains. The influence of the extraction methods on the composition, size diversity, molar mass and size distributions of exopolymeric substances (EPS) from the bacterium Sinorhizobium meliloti wild type (WT) and by the exoY strain deficient in exopolysaccharide production was investigated. EPS obtained by centrifugation, EDTA and formaldehyde/NaOH were compared. It was found that the extraction method influenced TOC, TN and total protein content in EPS from both strains. However, no difference between EDTA and formaldehyde/NaOH methods was observed for the exopolysaccharide components. Similar functional groups and fluorescence pattern were found in the EPS obtained by different methods; however their relative abundance was method dependent. The extraction method also affected the molar mass and size distribution, HP SEC diversity among different treatment and bacterial strains.