Background Grain chalkiness is one of important factors affected rice grain quality. It is known that chalkiness is affected by the high temperature during the seed filling period. Although a larger of QTLs for chalkiness were reported across all 12 chromosomes, only a few of the QTLs were fine mapped or cloned up to now. Here, we fine map two QTLs for chalkiness in two single-segment substitution lines (SSSLs), 11-09 with substitution segment from O. sativa and HP67-11 with substitution segment from O. glaberrima. Results The grain chalkiness of SSSLs 11-09 and HP67-11 was significantly lower than that in the recipient Huajingxian 74 (HJX74) in consecutive 8 cropping seasons. The regression correlation analysis showed that percentage of chalky grain (PCG) and percentage of chalky area (PCA) were significantly and positively correlated with percentage of grain chalkiness (PGC). Two QTLs for grain chalkiness were located on two chromosomes by substitution mapping. qPGC9 was mapped on chromosome 9 with an estimated interval of 345.6 kb. qPGC11 was located on chromosome 11 and delimited to a 432.1 kb interval in the O. sativa genome and a 332.9 kb interval in the O. glaberrima genome. qPGC11 is a QTL for grain chalkiness from O. glaberrima and was mapped in a new region of chromosome 11. The effect of two QTLs was incomplete dominance. The additive effects of two QTLs on chalkiness in second cropping season (SCS) were significantly greater than that in first cropping season (FCS). Conclusions qPGC11 is a new QTL for grain chalkiness. The two QTLs were fine mapped. The donor alleles of qPGC9 and qPGC11 were sensitive to the high temperature of FCS.