Abstract: In synthetic biology of cyanobacteria, unmarked deletions of large fragments accelerate the process of genome simplification. In this study, three genomic regions of more than 10 kb, namely Synpcc7942_0050-Synpcc7942_0064, Synpcc7942_0233- Synpcc7942_0253 and Synpcc7942_1391-Synpcc7942_1400, were successfully deleted in Synechococcus elongatus PCC 7942 via traditional homologous double crossovers. Non-replicative plasmids with the two flanking homologous fragments were constructed and introduced into cyanobacterial cells to produce single-crossover recombinants. At this step, single crossover recombination occurred in one or both homologous region(s). Next, colonies freed of sacB (a conditional lethal gene in the vector portion) were selected on sucrose plates. Such colonies could be either double-crossover recombinants or revertants, depending on where the second recombination occurred; however, those single crossover recombinants with the plasmid integrated at both homologous regions were inclined to produce mutants rather than revertants. Large fragment deletion mutants were then identified by PCR examinations. This study demonstrated that traditional homologous recombination techniques can be used to generate unmarked large fragment deletions in cyanobacterial genomes.