Species of the genus Daphnia Mueller, 1785, are ancient aquatic organisms with origins dating back to approximately 145 million years. Over time, geographical, ecological, and behavioral barriers have led to the diversification of these species. The mechanism of Daphnia survival and dispersal is predetermined by the capability of their ephippium-protected embryos to remain dormant for extended periods of time, thus facilitating their widespread dispersal by wind, water currents, and animal vectors. This paper presents the analysis of the Daphnia species diversity in the Caucasus region, specifically Georgia, conducted using mitochondrial 16S rDNA and cytochrome oxidase subunit I (COI) gene fragments. The phylogenetic analysis reveals distinct evolutionary lineages among Daphnia species, molecular clock estimates suggesting the early Miocene divergence pattern, followed by the glaciation events of the Pleistocene and the uplift of the Caucasus mountains. This research highlights the challenges that species-level identification represents, emphasizing the necessity for using multiple gene fragments for accurate identification. The findings provide unprecedented insights into the evolutionary history, dispersal mechanisms, and genetic identification of Daphnia species in the Caucasus region. These results contribute to the comprehensive understanding of the ecological role and adaptive strategies of Daphnia, with implications for biodiversity conservation and environmental monitoring in aquatic ecosystems.