The ability of various pests and diseases to adapt to a single plant resistance gene over time leads to loss of resistance in transgenic rice. Therefore, introduction of different pest and disease resistance genes is critical for successful cultivation of transgenic rice strains with broad-spectrum resistance to multiple pathogens. Here, we produced resistance rice lines with multiple, stacked resistance genes by stacking breeding and comprehensively evaluated their resistance toChilo suppressalis(striped rice stemborer),Magnaporthe oryzae(rice blast), andNilaparvata lugens(brown planthopper) in a pesticide-free environment.CRY1CandCRY2Aare exogenous genes fromBacillus thuringiensis.Pib,Pikm,andBph29are natural genes in rice. CH121TJH was introduced intoCRY 1C,Pib,Pikm,andBph29. CH891TJH and R205XTJH were introduced intoCRY 2A,Pib,Pikm,andBph29. Compared with those observed in their recurrent parents, CH121TJH significantly increased the mortality of borers. The other two lines CH891TJH and R205XTJH are the same result. Three lines introduction ofPibandPikmsignificantly reduced the area of rice blast lesions, and introduction ofBph29significantly reduced seedling mortality fromN. lugens. Introduction of the exogenous genes had relatively few effects on agronomic and yield traits of the original parents. These findings suggest that stacking of rice resistance genes through molecular marker-assisted backcross breeding can confer broad spectrum and multiple resistance in differently genetic backgrounds.