Chromosome segment substitution lines (CSSLs) are powerful tools to combine naturally occurring genetic variants with favorable alleles in the same genetic backgrounds of elite cultivars. An elite CSSL Z322-1-10 was identified from advanced backcrosses between a japonica cultivar Nipponbare and an elite indica restorer Xihui 18 by SSR marker-assisted selection (MAS). The Z322-1-10 line carries five substitution segments distributed on chromosomes 1, 2, 5, 6 and 10 with an average length of 4.80 Mb. Spikilets per panicle, 1000-grain weight, grain length in the Z322-1-10 line are significantly higher than those in Nipponbare. Quantitative trait loci (QTLs) were identified and mapped for nine agronomic traits in an F3 population derived from the cross between Nipponbare and Z322-1-10 using the restricted maximum likelihood (REML) method in the HPMIXED procedure of SAS. We detected 13 QTLs whose effect ranging from 2.45% to 44.17% in terms of phenotypic variance explained. Of the 13 loci detected, three are major QTL (qGL1, qGW5-1 and qRLW5-1) and they explain 34.68%, 44.17% and 33.05% of the phenotypic variance. The qGL1 locus controls grain length with a typical Mendelian dominance inheritance of 3:1 ratio for long grain to short grain. The already cloned QTL qGW5-1 is linked with a minor QTL for grain width qGW5-2 (13.01%) in the same substitution segment. Similarly, the previously reported qRLW5-1 is also linked with a minor QTL qRLW5-2. Not only the study is important for fine mapping and cloning of the gene qGL1, but also has a great potential for molecular breeding.