Pakistan is amongst the top five milk producing countries of the world but yet the introduction of value added dairy products is lacking. A three year research project (2006‐09) was planned and initiated in Dairy Technology Laboratory of National Institute of Food Science and Technology (NIFSAT), University of Agriculture, Faisalabad‐Pakistan to assess the effect of milk sources (cow and buffalo), starter cultures (indigenous and commercial) and milk fat levels (1.5% and 2.5%) and ripening period on different physico‐chemical characteristics of Mozzarella cheese. Standardized buffalo milk contained more protein (3.71%), casein (2.85) and lactose (4.88%) content as compared to the mixture of cow and buffalo milk protein (3.53), casein (2.79) and lactose (4.79%). The pH and acidity of all milk samples varied from 6.62 to 6.64 and 0.13 to 0.14%, respectively. The calcium content of buffalo milk was higher than that of mixture milk. Cheese prepared from these milk sources and was analyzed for composition, mineral contents, organic acid, functionality, sensory and proteolysis at prescribed intervals. Buffalo milk cheese contained significantly higher protein, ash, Na, Ca and P content but contained significantly lower moisture content than cheese from mixture milk. There was more firmness in buffalo milk cheese than mixture milk cheese. The milk fat level significantly affected the cheese chemical composition. The moisture, protein, Ca and P content observed higher in low fat milk cheese as compared to high fat milk cheese. Mozzarella cheese prepared from locally isolated starter cultures possessed significantly higher moisture and acidity content, as compared to commercial culture cheese. The protein and pH were lesser in cheese that is primed from indigenous culture as compared to commercial culture. The effect of culture on fat, ash, salt and mineral contents was found to be nonsignificant. All the organic acids were significantly affected by the ripening periods. The fat levels significantly effected the concentration of lactic and citric acid but the formic and pyruvic acid was found to be non‐significant due to milk fat level. The functional properties (free oil, flowability, stretchability and melt time) of Mozzarella cheese were significantly affected by the ripening period and fat level. The free oil formation and flowability increased from 20.35% to 29.99% and 17.63% to 48.10%, respectively during ripening period. The stretchability of cheese first increase up to 40 days of ripening (35.31 to 49.59 cm) and then decreased (43.75 cm) at 60 day. The low fat cheese had lower free oil 15.74% and flowability 29.81% as compared to high fat cheese. The stretchability is more in low fat cheese (47.15cm) in comparison to high fat cheese (40.41cm). The low fat milk cheese took more time to melt (135.8 sec) as compared with high fat milk cheese (119.8 sec). The expressible serum was significantly affected by the ripening period and fat levels. There was a decrease in expressible serum from 22.63g/100g to 3.85g/100g during ripening. The serum was recorded more in high fat cheese as compared to low fat cheese. Similarly low fat cheese was more firm (332.50 N) than high fat cheese (284.85 N) and commercial cultured cheese was relatively more firm (312.36N) as compared to indigenous cultured cheese (304.39N). The assessment of sensory texture of Mozzarella cheese during the ripening exhibited that all the cheese samples became significantly less firm over time as indicated by sensory measurement. All the sensory parameter such as hand firmness first bite firmness, chewdown degree of break down , and chew down smoothness 54 were significantly affected by the fat levels in milk but the other factor such as milk source and the starter culture did not affect these parameters significantly. There was a decrease in extent of blistering with the progressive ripening and increase in flavour, oiling and shred fusion was observed.