Advances in plant molecular techniques have dramatically widened the applicability of gene identification and pyramiding valuable genes. This study was carried out to pyramid five resistance genes for biotic stress into the japonica rice cultivar using marker-assisted selection (MAS) and marker-assisted background analysis of selected progenies using SSR markers. The Pi40, Xa4, xa5, Xa21 and Bph18 genes were combined in Jinbubyeo, a Korean japonica rice variety using MAS. Gene specific co-dominant PCR-based markers were used to select for homozygous recombinant lines in a segregating population derived from a cross between the parental homozygous resistant gene introgression lines. We had successfully developed multiple gene pyramided breeding lines (GPLs) for bacterial blight, blast, and brown planthopper using MAS in rice. The GPLs exhibited high resistance against biotic stress and had around 93% of the genetic background of the recurrent parent Jinbubyeo based on SSR graphical mapping. The yield and agronomic traits of the GPLs were similar to those of the recurrent parent, indicating that there is no apparent agronomic trait penalty associated with the presence of the resistance genes. The strategy of simultaneous foreground and phenotypic selection to introduce multiple R genes is very useful to reduce the cost and the time required for the isolation of desirable recombinants with target resistance genes in rice. The GPLs could be useful to enhance effective resistance for biotic stress and produce stable grain yield in japonica rice breeding programs.

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