Two different cDNA clones for the chloroplast small heat shock protein (smHSP) were isolated from tobacco (Nicotiana tabacum cv. Petit Havana SR1). One of the cDNAs (type I) has a full-length open reading frame (ORF) of the smHSP of 26.6 kDa. By contrast, the other one (type II) contains an additional nucleotide that causes the frame shift inside a putative ORF for the smHSP. If this nucleotide is neglected, type II cDNA encodes the smHSP that is 89% identical to that encoded by type I cDNA. Southern blot and polymerase chain reaction (PCR) analyses with genomic DNA indicated that tobacco has two different smHSP genes while two ancestors of tobacco, N. sylvestris and N. tomentosiformis, have a single gene that each corresponds to one of the two genes of tobacco. It was also found that one of the tobacco genes has an ORF for the smHSP disrupted by nucleotide insertion in the same way as type II cDNA, while both ancestor genes have a functional ORE These results suggest that the two smHSP genes of tobacco had been derived from the two ancestor species, and that one of the two genes had been disrupted by nucleotide insertion during the course of the evolution of tobacco. Northern blot and reverse transcription (RT)-PCR analyses demonstrated that both the tobacco genes are expressed upon heat stress, exhibiting different dependences on temperature.