Excess ferrous iron in lowland soil is known to inhibit the growth of rice. A quantitative trait locus (QTL) analysis for susceptibility to ferrous iron was performed using chromosomal segments substitution lines (CSSLs). Kasalath, an indica rice cultivar, is known to be susceptible to ferrous iron and accumulate excess iron in shoots. The shoot iron concentration was examined in 39 CSSLs carrying Kasalath chromosomal segments in a background of Koshihikari, a japonica cultivar. Kasalath grown in a hydroponic culture solution containing excess ferrous iron, had a higher shoot iron concentration than Koshihikari. Of the CSSLs, SL208, which carries the Kasalath chromosomal segment on chromosome 3, had a significantly higher shoot iron concentration than Koshihikari, and none of the CSSLs had a shoot iron concentration significantly lower than Koshihikari. This finding suggests that the putative QTL affecting the shoot iron concentration is between the markers R663 and S1571 on chromosome 3.