Heat shock transcription factor 1 (HSF1) mediates the induction of heat shock proteins in response to various types of stress. Although HSF1 activity is regulated by its post-translational modifications, alterations in mRNA expression have also been suggested. We here identified three new alternatively spliced isoforms of Drosophila HSF (dHSF) mRNA, named dHSFb, dHSFc, and dHSFd. We found that the ratio of dHSFb increases upon heat exposure, while that of dHSFd increases upon cold exposure. The dHSFc and dHSFd isoforms showed greater transcriptional activity than the other isoforms. Our findings suggest that alternative splicing regulates the transcriptional activity of dHSF.