Small heat-shock proteins (sHSP) are the most abundant heat stress-induced proteins in plants. In rice, there are at least seven members of class-I sHSP. A 1.6-kb DNA fragment was isolated from the EcoRI-digested rice genomic library probed with the cDNA pTS1 encoding a 16.9-kDa class-I sHSP. This fragment was composed of 365-bp tandem direct repeats (DRs) and 441-bp near perfect long terminal inverted repeats (LTIRs). The DRs contain 123-bp regions with 99% nucleotide identity to the 5' coding region of the Oshsp16.9B gene. Two putative pseudogenes were deduced from the DRs. Using the LTIR as a specific probe, Southern-blotting analysis showed that there was a single copy of this 1.6-kb DNA fragment in the rice genome. By genomic walking, we located this fragment in proximity 5'-upstream of the Oshsp16.9B gene that was mapped on chromosome 1 with other two class-I sHSP genes, Oshsp16.9A and Oshsp16.9C. By comparative analysis of the nucleotide sequences of class-I sHSP genes clustered on chromosome 1 between Tainung No. 67 and Nipponbare cultivars, we confirmed our mapping results of these genes and only the promoter region of Oshsp16.9B was different. However, we found that the expression profile of Oshsp16.9B upon different heat stresses in Nipponbare was not significantly different relative to that in Tainung No. 67.