Culture collections contain cyanobacterial and microalgal strains as type species taxonomical studies and future exploitation in basic and applied research. The Culture collection of experimental strains, isolated from cold environments, at the Institute of Botany CAS, Třeboň, Czech Republic, is a working non-public culture collection, i.e., the strains are being isolated and used in experiments, but they are not available for sale. At present (June 2025), the culture collection contains 350 defined strains of cyanobacteria and microalgae isolated from Antarctica (168 strains), Arctic (148 strains), Europe (18 strains) and North America (14 strains). The origin of two strains is unknown. The strains originate from snow/glacier (17 strains), lacustrine (39 strains), hydro-terrestrial (24 strains), and terrestrial (72 strains) environments. It is planned to determine the original environment of the remaining 198 strains in the near future. From a taxonomical point of view, 153 strains belong to the Cyanophyceae class, 38 to the Trebouxiophyceae class, 32 to the Chlorophyceae class, 23 to the Klebsormidiophyceae class, 8 to the Zygnematophyceae class and 94 to the Xanthophyceae class. The Ulvophyceae and Bacillariophyceae classes are both represented by a single strain. In addition, methods of sampling, isolation, maintenance, and determination of strains are described.