Over 7 individual rice (Oryza sativa L.) plants per a line were sowed and sampled by pooled sampling method for genomic DNA extraction. The 5,400 flanking sequence tags (FSTs) were analysed by adaptor PCR and direct sequencing. FST analysis showed that the intragenic FSTs, the intergenic FSTs, and the original insertional sequences including hot spot covered 48.1% (2,597), 25.6% (1,383), and 25% (1,350), respectively. The 2,597 intragenic FSTs were used for genotyping to determine whether they are heterozygous or homozygous, and 1,393 core lines were selected. Among them, 422 knockout genes were distributed on chromosome 3, while 56 - 157 intragenic FSTs scattered on other chromosomes. Among 1,393 FSTs, known genes such as transcription factor covered 59.4% (827), while unknown genes such as expressed protein covered 40.6% (566). RT-PCR indicated that some core lines had no expression or decreased expression level in their knockout genes. It means that core lines are very useful knockout lines for functional genomic studies.