Most agronomic traits of rice ( Oryza sativa ), such as grain length, are complex traits controlled by multiple genes. Chromosome segment substitution lines (CSSLs) are ideal materials for dissecting these complex traits. We developed the novel rice CSSL ‘Z414’, which has short, wide grains, from progeny of the recipient parent ‘Xihui 18’ (an indica restorer line) and the donor parent ‘Huhan 3’ (a japonica cultivar). Z414 contains four substitution segments with an average length of 3.04 Mb. Z414 displays seven traits that significantly differ from those of Xihui 18, including differences in grain length, width, and weight; degree of chalkiness; and brown rice rate. We identified seven quantitative trait loci (QTL) that are responsible for these differences in an F2 population from a cross between Xihui 18 and Z414. Among these, six QTL ( qPL3 , qGW5 , qGL11 , qRLW5 , qRLW11 , and qGWT5 ) were detected in newly developed single-segment substitution lines (SSSLs) S1–S6. In addition, four QTL ( qGL3 , qGL5 , qCD3 , and qCD5 ) were detected in S1 and S5. Analysis of these SSSLs attributed the short, wide grain trait of Z414 to qGL11 , qGL3 , qGL5 , and qGW5. Substitution mapping delimited qGL11 within an 810-kb interval on chromosome 11. Sequencing, real time quantitative PCR, and cell morphology analysis revealed that qGL11 might be a novel QTL encoding the cyclin CycT1;3. Finally, pyramiding qGL3 (a = 0.43) and qGL11 (a = − 0.37) led to shorter grains in the dual-segment substitution line D2 and revealed that qGL11 is epistatic to qGL3 . In addition, S1 and D2 exhibited different grain sizes and less chalkiness than Z414. In conclusion, the short grain phenotype of the CSSL Z414 is controlled by qGL11 , qGL3 , and qGL5 . qGL11 might be a novel QTL encoding CycT1;3, whose specific role in regulating grain length was previously unknown, and qGL11 is epistatic to qGL3 . S1 and D2 could potentially be used in hybrid rice breeding. Supplementary Information The online version contains supplementary material available at 10.1186/s12284-022-00571-7.