Plant heat shock transcription factors (HSF) are highly complex. In this study, we identified an alfalfa HSF gene MsHSF1 that is composed of four exons and three introns in the encoding region. The intron1-exon2-intron2-exon3-intron3 as an intervening sequence was inserted at the conserved position that separates the coding region for the DNA-binding domain by single intron in other known plant HSF genes. Alternative splicing of MsHSF1 has generated five transcript isoforms. Spliced transcript MsHSF1b consisted of exon1 and exon4, encodes a class A1 HSF protein that can specifically bind to the heat shock elements in vitro. Other four spliced transcripts (MsHSF1a-1 to 4) consist of exon1, part of the intervening sequence and exon4. These transcripts carry the premature termination codon and are low-abundant. Apparently these transcripts are the targets of nonsense-mediated mRNA decay (NMD). These results provide new insight into roles in the expression regulation of plant HSF genes.