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Rice chromosomal segment substitution lines (CSSLs) are ideal materials for studying quantitative traits such as grain size. Here, a rice large-grain CSSL-Z403 was identified among progeny of the recipient Xihui18 and the donor Jinhui35 based on molecular marker-assisted selection. Z403 carried 10 substitution segments with average length of 3.01 Mb. Then, a secondary F₂ population derived from a cross between Xihui18 and Z403 was used to map quantitative trait loci (QTL) for grain size. Six QTLs distributed on chromosomes 5, 6, 7, 9 and 12 were detected. Finally four single-segment substitution lines (SSSLs) and two dual-segment substitution lines (DSSLs) carrying these target QTLs were constructed, and 10 novel QTLs were identified by four SSSLs. The large grain of Z403 was controlled at least by <i>qGWT5</i>, <i>qGWT7</i>, <i>qGWT9</i> and <i>qGWT12</i>, and its grain weight was influenced through grain length QTL such as <i>qGL5</i>, <i>qGL6</i>, <i>qGL9</i> and <i>qGL12</i>, as well as grain width QTL such as <i>qGW5</i>, <i>qGW7</i>, <i>qGW9</i> and <i>qGW12</i>. Among 16 QTLs, four QTLs including <i>qGL6</i>, etc., might be novel compared with the reported documents. Again, positive or less negative epistatic effects between two non-allelic QTLs (additive effect > 0) may assist screening the genotype with larger grain size in further selection.