Heterogeneity of glia in different CNS regions may contribute to the selective vulnerability of neuronal populations in neurodegenerative conditions such as amyotrophic lateral sclerosis (ALS). Here, we explored regional variations in the expression of heat shock protein 25 in glia under conditions of acute and chronic stress. Hsp27 (Hsp27; murine orthologue: Hsp25) fulfils a number of cytoprotective functions and may therefore be a possible therapeutic target in ALS. We identified a subpopulation of astrocytes in primary murine mixed glial cultures that expressed Hsp25. Under basal conditions, the proportion of Hsp25‐positive astrocytes was twice as high in spinal cord cultures than in cortical cultures. To explore the physiological role of the elevated Hsp25 expression in spinal cord astrocytes, we exposed cortical and spinal cord glia to acute stress, using heat stress and pro‐inflammatory stimuli. Surprisingly, we observed no stress‐induced increase in Hsp25 expression in either cortical or spinal cord astrocytes. Similarly, exposure to endogenous stress, as modelled in glial cultures from SOD1G93A‐ALS mice, did not increase Hsp25 expression above that observed in astrocytes from wild‐type mice. In vivo, Hsp25 expression was greater under conditions of chronic stress present in the spinal cord of SOD1G93A mice than in wild‐type mice, although this increase in expression is likely to be due to the extensive gliosis that occurs in this model. Together, these results show that there are differences in the expression of Hsp25 in astrocytes in different regions of the central nervous system, but Hsp25 expression is not upregulated under acute or chronic stress conditions.