The fine structure of amylopectin affects rice quality; in particular, the amylopectin chain length distribution (ACLD) in milled rice differs between subspecies of Oryza sativa L. However, the correlation between ACLD and quality trait factors, and the genetic basis of ACLD phenotypic variation, are still unknown. Here, the correlations of ACLD with cooking and eating quality and with the rapid viscosity analysis (RVA) index were studied using chromosome segment substitution lines (CSSLs). Clear variations in ACLD were observed in introgression lines: introgression of indica segments of chromosome 3 and 7 increased the proportion of amylopectin Fa, and another segment of chromosome 3 reduced the proportion of amylopectin Fb2. A segment of chromosome 11 decreased the proportion of amylopectin Fa but increased that of Fb3. Correlation analysis with the RVA index further showed that the breakdown viscosity (BDV) was negatively correlated with the proportion of amylopectin Fb1, Fb2, and Fb3 chains, and positively correlated with Fa. Consistency viscosity (CSV) values were negatively correlated with the proportion of amylopectin Fb1, Fb2, and Fb3 chains. We thus clarified the quality trait factors determined by variation in ACLD, and provide key information for pyramiding inter-subspecific genetic superiority in molecular design breeding for rice quality.