This study was conducted to develop multi-resistant lines to brown planthopper, bacterial blight, and rice stripe virus using anther culture in rice. A total of 213 double haploid lines were developed the cross between HR26234-12-1-1 conferring resistant to bacterial blight and rice stripe virus and SR30071-3-7-23-6-2-1-1 conferring resistant to brown planthopper, bacterial bight, and rice stripe virus. Using DNA molecular marker, HR26234 and SR30071 were confirmed to have Xa3+xa5+Stvb-i and Bph18+Xa4+Stvb-i, respectively. All double haploid lines carried Stvb-i, and Bph18+Xa3, Bph18+Xa4, Bph18+Xa3+xa5, Bph18+Xa4+xa5, bph18+Xa3, bph18+Xa4, bph18+Xa3+xa5, and bph18+Xa4+xa5 combinations were identified. Segregation distortions such as no combinations carrying Bph18(or bph18)+xa5+Stvb-i and fewer lines carrying Bph18 than bph18 were occurred in DH population. Brown planthopper resistant lines carrying Bph18 showed longer culm length than susceptible lines. Selected Bph18+Xa4+xa5+Stvb-i combination lines with short culm conferred resistant to brown planthopper, bacterial blight, and rice stripe virus, while showed deleterious effects such as spikelet sterility, lower yield, and vulnerable to lodging than standard and comparative varieties. Using anther culture, we rapidly developed multi-resistant lines to brown planthopper, bacterial blight, and rice stripe virus. However, distorted segregation in DH population and linkage drag with Bph18 were obstacles to develop practical multi-resistant cultivars.