The effects of two quantitative trait loci (QTLs) controlling the resistance to rice stripe virus (RSV) identified in a Japanese upland rice line, Kanto 72, (URK 72) were evaluated using near-isogenic lines (NILs). Two NILs carrying a single QTL (QTL-NILs) on chromosomes 2 and 11, respectively, were developed by marker-assisted selection (MAS). The target QTL regions were introduced from the donor parent, Chugoku 40, which was bred from URK 72, into the genetic background of Koshihikari. Another line, which was a combined QTL-NIL, was developed from the cross between the two QTL-NILs to analyze the interaction of the two QTLs. Investigation of RSV resistance using the three NILs revealed that the effects of the two QTLs clearly differed in the reaction to RSV. The QTL on chromosome 11 exerted a major effect on reducing the infection rate of RSV. Although the QTL on chromosome 2 did not affect the infection rate, the symptoms of the diseased plants were milder. The combined QTL-NIL displayed a high level of resistance to RSV, while the infection rate and the symptom types of the diseased plants were similar to those of URK 72 or Chugoku 40. Since the major agronomic characters of the three QTL-NILs were the same as those of Koshihikari, these QTL-NILs were considered to be useful gene sources for RSV resistance rice breeding.