Although polar regions are characterised by extreme environment conditions with overall low temperatures and often pronounced diurnal to seasonal temperature fluctuations, diatoms (Bacillariophyceae) thrive in a wide range of nonmarine polar habitats, suggesting that they are well adapted to these harsh conditions. In this study, tolerance to freezing of vegetative and resting cells of 17 polar and nine temperate diatom strains, belonging to different morphospecies and originating from both freshwater and terrestrial habitats, was experimentally tested. Most strains were newly isolated from sites in Maritime Antarctica (James Ross Island and Vega Island), the High Arctic (Spitsbergen) and Europe. Cultures were exposed to five freezing treatments differing in temperature (−4, −20, −40 and −180 °C), freezing rate (gradual, abrupt), time (1 h, 12 h) and thawing rate (slow, fast). Results indicated that diatoms were sensitive to experimental freezing. Freezing temperatures had a significant effect on strain survival: all strains survived −4 °C; most survived −20 °C; five survived −40 °C, and four of these (all belonging to the Pinnularia borealis complex) survived freezing in liquid nitrogen (−180 °C). The cooling and thawing rate had a significant impact on survival: abrupt cooling and slow thawing resulted in much lower survival rates than gradual cooling and fast thawing. Resting cells showed better growth than vegetative cells after freezing treatment but only in the −4 °C treatment. Surprisingly, no striking differences in growth recovery were observed between polar and temperate strains.