Synechocystis sp. PCC 6803 is the most popular cyanobacterial strain, serving as a standard in the research fields of photosynthesis, stress response, metabolism and so on. A glucose-tolerant (GT) derivative of this strain was used for genome sequencing at Kazusa DNA Research Institute in 1996, which established a hallmark in the study of cyanobacteria. However, apparent differences in sequences deviating from the database have been noticed among different strain stocks. For this reason, we analysed the genomic sequence of another GT strain (GT-S) by 454 and partial Sanger sequencing. We found 22 putative single nucleotide polymorphisms (SNPs) in comparison to the published sequence of the Kazusa strain. However, Sanger sequencing of 36 direct PCR products of the Kazusa strains stored in small aliquots resulted in their identity with the GT-S sequence at 21 of the 22 sites, excluding the possibility of their being SNPs. In addition, we were able to combine five split open reading frames present in the database sequence, and to remove the C-terminus of an ORF. Aside from these, two of the Insertion Sequence elements were not present in the GT-S strain. We have thus become able to provide an accurate genomic sequence of Synechocystis sp. PCC 6803 for future studies on this important cyanobacterial strain.