In plants, small heat shock proteins (sHsps) have been localized to the cytosol, mitochondria, chloroplasts, and endoplasmic reticulum. Using NtHSP24.6, a cDNA clone for tobacco mitochondrial sHsp, as a probe, we performed genomic DNA blot analysis and identified presence of a small gene family for mitochondrial sHsp in tobacco (Nicotiana tabacum). Several putative genomic clones for mitochondrial sHsp were isolated when a tobacco genomic library was screened. After restriction mapping of seven genomic clones, three that had shown different maps were selected, and their nucleotide sequences for the putative coding sequence were determined. From these three independent clones, one identical nucleotide sequence was obtained that had two exons with one intron. RNA blot hybridization of heat-stressed tobacco plants revealed a typical heat-shock-responsive accumulation of NtHSP24.6 transcript. Under severe heat-shock conditions, an additional band was apparent, but of a larger transcript size. When we compared the amino acid sequence of NtHSP24.6 with mitochondrial sHsps from various other species, we found a high level of homology throughout the ORF, with an almost complete match to the carboxyl terminus. Our comparison of NtHSP24.6 with tobacco cytosolic class I sHsps also resulted in high homology between their $alpha$-crystalline domains, but significant divergence in their amino-terminus regions.