A novel population consisted of 86 single segment substitution lines (SSSLs) were developed from advanced backcrosses between the recipient, Huajingxian 74 and six donors by microsatellite marker-assisted selection (MAS). Fifty-two SSSLs were selected in BC3F2, and 34 others were selected in BC3F3. Every SSSL contains a single chromosome segment introgressed from one donor on the genetic background of Huajingxian 74. The substituted segments in SSSLs were distributed on 12 rice chromosomes. The estimated length of the substituted segments in SSSLs ranged from 1.5 cM to 56.3 cM with an average of 23.0 cM. Total of the substituted segments covered 57.1% of the rice genome.