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Cytoplasmic male sterility (CMS) is an important foundation for heterosis utilization andexploited widely to hybrid seed production in rice.However, the traditional experiencedriven and labor-cost breeding style left little room for sustainable improvement ofhybrid rice.Development of molecular biology and genomics provides a chance toenhance hybrid rice breeding efficiency through breeding by design.In this study, aline H121B with rf3 and rf4 genes from XieqingzaoB (XB) was developed in theHuajingxian74 (HJX74) genetic background.Using H121B as maintainer line, threeisonuclear alloplasmic CMS lines, W-H121A, D-H121A and Y-H121A, with the wildabortive (WA), dwarf wild abortive (DA), and Yegong abortive (YA) types of CMScytoplasm, respectively, were developed.The H121B maintainer line was thenimproved by a way of breeding by design.Three favorable alleles, gs3 for long grain, Wxtfor low amylose content, and OsMADS50 for early heading date, in the chromosomalsubstituted segments of single segment substitution lines (SSSLs) were selected fromthe SSSL library in the HJX74 genetic background, and were pyramided together inH121B through marker-assisted selection (MAS).The new maintainer line, H131B,showing the phenotypes of longer in grain length, lower in amylose content, and earlierin heading date than those in H121B, achieved the breeding goal designed.UsingH131B as maintainer line, three new isonuclear alloplasmic CMS lines, W-H131A, DH131A and Y-H131A, were developed.Those results indicate that breeding by designin rice can be conducted under the platform of the HJX74 SSSL library.Establishmentof the platform for breeding by design of CMS lines will be helpful for sustainableimprovement of CMS lines in hybrid rice development.