Heat shock proteins or chaperones are found in mitochondrial and cytosolic compartments of cells. They are responsible for the correct folding of proteins and are up-regulated in reaction to various stressors. In addition, when released or presented on the surface of cells, they may play an important role in inflammatory and immunomodulating processes. To identify and characterize hsp60 in the common environmental mold Alternaria alternata, the fungus was cultivated and incubated at different temperatures to induce a possible heat shock response. Fully automated RNA extraction was followed by quantitative real-time RT-PCR targeting A. alternata specific Hsp60 mRNA and subsequent sequencing of the amplicon. While Hsp60 mRNA was constitutively expressed in all samples tested, a temperature-dependent expression of Hsp60 mRNA was observed. Sequencing revealed an identity of more than 85% to other fungal hsp60, indicating the existence of this protein in A. alternata.