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The dairy industry releases huge amounts of by-products. One of them is buttermilk, obtained during butter production. This by-product is characterized by high nutritional and technological value and is finding more and more applications in food production. This study aimed to produce and analyze the characteristics of quark cheese obtained entirely from buttermilk during 3-week refrigerated (4 ± 1 °C) storage. Four kinds of sour buttermilk were used: two from industrial butter production, and another two from butter production at laboratory scale. Laboratory buttermilk differs in the kind of starter culture used in the production. The evaluation of cheese quality properties included physicochemical analyses, texture measurement, and sensory assessment. The results showed that the kind of buttermilk used in production influences the acidity, total solids, textural characteristics, and fat content of the obtained quark cheeses. All obtained cheeses had very high sensory quality throughout the storage period. The study indicates that buttermilk may be successfully used as a substitution for milk in quark cheese production.