Chalkiness is one of the important traits affecting milling quality and appearance of rice grains. The purpose of this study was to identify quantitative trait loci (QTL) associated with grain chalkiness, and select rice superior lines with less grain chalkiness by marker assisted selection (MAS). A total of 840 lines derived from a cross between rice varieties Koshihikari and TNG67 were used as materials. Among these, 107 F2 derived lines were used to build a linkage map composing a total of 112 SSR and InDel polymorphic DNA markers. Phenotypic data of these 107 lines were used to identify QTL for three grain chalkiness related traits, percentage of grains with chalkiness (PGWC), area of chalky endosperm (ACE) and degree of endosperm chalkiness (DEC). QTL of plant height (PH), days to heading (HD) and panicle number (PN) were also identified by interval mapping analysis. According to the QTL mapping result, three markers, RM1248, CH0701 and RM6306, associated with grain chalkiness and one marker, RM4108, associated with eating quality trait were used to assist selecting 23 lines with less grain chalkiness. Twenty-two of the selected lines had better grain chalkiness trait than the referenced parent TNG67, confirming the expectation of MAS effects. The selected F2:5 lines were genetically purified with single plant selection based on marker genotypes of segregating loci correlated with chalkiness grain. According to the grain chalkiness and agronomic traits investigated under different environments, the most promising lines will be recommended. In this study, because MAS for the improvement of grain chalkiness traits was applied at the early generation (F2 generation), the labor and time resources were saved from the traditional breeding procedures, and the environmental disturbance for selection could be avoided. The results of this study can provide information for enhancing rice breeding efficiency.