The Ancylonema genus includes the most-documented microalgae on glaciers and ice sheets worldwide. There is significant interest in these microalgae in the context of climate change, considering their role in lowering surface ice albedo and acceleration of ice melt. However, currently, no cultures of the two closely related species A. nordenskiöldii or A. alaskanum have been established, restricting our ability to study these globally important species under laboratory conditions. We established and kept cultures of Ancylonema sp. alive for up to 2 years, by testing and optimizing different growth media and parameters. Maximum growth was achieved when using 1:100 diluted media with soil extract and low light intensity (300 µmol m−2s−1). However, as a consequence of incubation in lab conditions, some of the cultures lost their purpurogallin pigmentation and appeared green. Sanger sequencing of the ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit (rbcL) marker gene revealed a large genetic diversity (possibly cryptic) and confirmed the cultures as falling within the same clade as A. nordenskiöldii and A. alaskanum. Growth experiments allowed us to estimate a division rate of between 15 ± 5.2 and 21.9 ± 4.8 days. This is up to 4 times slower than current field-based estimates (3.75–5.5 days) and indicates that, despite the successful growth and long-term maintenance of the cultures, laboratory settings can be further improved to achieve optimal growth conditions.