Thermal springs represent extreme ecological niches characterized by elevated temperatures and mineral-rich waters, shaping highly specialized microbial communities. Cyanobacteria, as primary producers in these habitats, exhibit remarkable adaptations, yet their diversity and phylogenetic relationships in Serbian geothermal systems remain poorly documented. In this study, we applied a molecular systematics approach based on 16S rRNA gene sequencing to characterize cyanobacterial assemblages from five thermomineral springs in Serbia. Field sampling was conducted during October and November, and all strains were subsequently isolated and cultivated in BG11 medium for several months to ensure stability and purity prior to molecular analysis. The molecular analyses also facilitated the confirmation of morphological identification. Leptolyngbya sp. (WUC607) was detected in Niška Banja (38–39 °C), while Leptolyngbya sp. (CALU1715) was found in Lopatnica (≈29 °C) and Šarbanovac (30 °C). Wilmottia sp. (CCNU0051) was identified in Brestovačka Banja (35 °C). In Vranjska Banja (surface 93 °C), both Nodosilinea sp. (PGN35) and Anabaena variabilis ("Vasse River Type 3") were found. This study provides molecular characterization of cyanobacterial communities from Serbian thermal springs, enriching a knowledge in thermophilic microorganisms and potentially discovery of new species. The findings emphasize the value of molecular systematics for better understanding evolutionary adaptations to thermomineral environments. Moreover, these thermophilic lineages represent potential candidates for further research on thermal tolerance mechanisms and potential biotechnological applications, such as bioactive compound production.