Most strains of S.thermophilus do not ferment galactose and releases free galactose into the fermented milk and cheese. The inability of S. thermophilus to utilize galactose may be due to poor expression of one or more catabolic enzymes. Accumulation of free galactose in fermented dairy products leads to browning reactions in cheese, heterofermentation and galactosomeia in consumers. In milk fermentation, S. thermophilus AJM released less galactose (0.27%) followed by S. thermophilus KM3 (0.3%) and S. thermophilus JM1 (0.45%). The gal-lac gene order (galRKTEM-lacSZ) was found to conserve and individual size of the gene are highly similar in both gal- and gal+ strains by Long Range PCR. But the promoter sequence of the gal+ isolates (AJM, JM1&KM3) possessed single base pair deletion at -35 region region of galR promoter and C to T substitution at -10 box of galK region. In contrast, AUKD8 has A to T substitution at preceding -10 box. Yoghurt prepared with gal+ isolate AJM contained less galactose 0.37% followed by KM3(0.51%) and JM1(0.65%) as compared to gal- NCDC 218(0.98%) after 4h of fermentation.