Abstract Since it has been observed that low temperature protein crystal structures may differ from room temperature structures, it is necessary to compare systematically the protein hydration structure in low and room protein crystal structures. High quality data sets of protein structures were built in an extremely rigorous manner and crystal symmetry was included in the identification of four types of water molecules (buried in the protein core, deeply inserted into crevices at the protein surface, first and second hydration layers). More water molecules are observed at low temperature only if the resolution is better than 2.1–2.3 Å. At worse resolution, temperature does not play any role. The numerous water molecules that become detectable at low temperature and at higher resolution are more mobile, relative to the protein average flexibility. Despite that, the occupancy does not depend on temperature. It can be hypothesized that water structure and around proteins and hydrogen bond network do not depend on the temperature, at least in the temperature range examined here. At low temperature more water molecules are detected because the average flexibility of all the atoms decreases, so that also water molecules that are considerably more mobile than the average atoms become observable in the electron density maps.