Abstract Aims Although kefir has been known for centuries, there is confusion between the two types of kefir grains, for example, milk kefir (MK) grain and water kefir (WK) grain. This study aimed to unravel the differences and similarities between WK grain and MK grain. Methods and Results Microbiological analyses, identification of grains microbiota and enumeration of microbiological content of the grains as well as scanning electron microscope (SEM) imaging, dry matter, protein, ash, and mineral content, and colour analyses were carried out for the two types of grains. As a result, significant differences were found in microbiological content, chemical properties and colours (p < 0.05). Additionally, SEM images revealed the different intrinsic structures for the microbiota and the structure of the two types of grains. Conclusions MK grain has more nutritional content compared to WK grain. Despite not as widely known and used as MK grain, WK grain is a good source for minerals and health-friendly micro-organisms such as lactic acid bacteria and yeasts. WK grain is possibly suitable for vegans and allergic individuals to fulfil nutritional requirements. Moreover, in this study, the variety of WK grain microbial consortia was wider than that of MK grains, and this significantly affected the resultant WK products. Significance and Impact of the Study This is the first study that comprehensively compares two different kefir grains in microbial, chemical and physical properties.