Granule cell dispersion (GCD) has been found in the dentate gyrus (dg) of patients with temporal lobe epilepsy (TLE) and a history of febrile seizures but was also recently observed in pediatric patients that did not suffer from epilepsy. This indicates that GCD might not always be disease related, but instead could reflect normal morphological variation. Thus, distribution of newborn granule cells within the hilar region is part of normal dg development at early stages but could be misinterpreted as pathological GCD. In turn, pathological GCD may be caused, for example, by genetic mutations, such as the reeler mutation. GCD in the reeler mutant goes along with an increased susceptibility to epileptiform activity. Pathological GCD in combination with epilepsy is caused by experimental administration of the glutamate receptor agonist kainic acid in rodents. In consequence, the interpretation of GCD and the role of febrile seizures remain controversial. Here, we asked whether febrile temperatures alone might be sufficient to trigger GCD and used hippocampal slice cultures as in vitro model to analyze the effect of a transient temperature increase on the dg morphology. We found that a heat-shock of 41°C for 6 h was sufficient to induce GCD and degeneration of a fraction of granule cells. Both of these factors, broadening of the granule cell layer (gcl) and increased neuronal cell death within the gcl, contributed to the development of a significantly reduced packaging density of granule cells. In contrast, Reelin expressing Cajal–Retzius (CR) cells in the molecular layer were heat-shock resistant. Thus, their number was not reduced, and we did not detect degenerating CR cells after heat-shock, implying that GCD was not caused by the loss of CR cells. Importantly, the heat-shock-induced deterioration of dg morphology was accompanied by a massive microgliosis, reflecting a robust heat-shock-induced immune response. In contrast, in the study that reported on GCD as a non-specific finding in pediatric patients, no microglia reaction was observed. Thus, our findings underpin the importance of microglia as a marker to distinguish pathological GCD from normal morphological variation.