Water kefir is a fermented probiotic beverage produced by fermentation of dried sugary fruits and sugar using water kefir grains. Sucrose is used as a sugar source in the traditional production of water kefir. The aim of this study is to determine the effects of grape molasses, honey and unrefined sugar on water kefir grains instead of sucrose. In the study, 4 groups were formed: grape molasses (10 g), honey (10 g), unrefined sugar (10 g) and refined sugar (10 g). 2 g of water kefir grain and 500 mL of drinking water were used for each group. And each group samples were fermented for 25°C 48 hours. The study continued for 5 weeks. The pH values and the titratable acidity values of water kefir samples and the weight of water kefir grains were determinated once every two day, while microbiological analyzes (Lactobacillus spp., Lactococcus spp. and yeast contents) and ethanol content of samples were determinate once every 15 days. In this study; it has been observed that the use of different sugars has no significant effect on the weights of the water kefir grains. In addition, it was determined that the Lactococcus spp. and yeast amounts in the water kefirs were higher in the samples produced by using honey and grape molasses. As a result, it was revealed that grape molasses, honey and unrefined sugar could be used as fermentation substrate in production of water kefir. At the same time, water kefir can be shown as an alternative fermented product for vegan consumers with allergies to dairy products.