Thousand grain weight (TGW), determined by grain size and grain filling, is a very important trait for yield improvement in wheat, which is less sensitive to environments with high heritability. In order to dissect the underlying genes and molecular mechanisms, 266 recombinant inbred lines (RILs) derived from a cross between Zhongmai 871 (ZM871) and its sister line Zhongmai 895 (ZM895) were evaluated for TGW in ten environments during 2014-2017 cropping seasons, and genotyped by the wheat 660K SNP array. Large variation of TGW was observed, ranging from 39.6 to 54.5 g in the RIL population. A stable QTL QTgw.caas-5B was identified in all the ten environments, explaining 5.2%-15.2% of the phenotypic variances with the favorable allele from ZM895. The QTL was further delimited into an interval of approximately 1.0 Mb flanked by markers Kasp-5B35 and Kasp-5B37 (50.3 to 51.3 Mb), using 12 heterozygous recombinant plants obtained by selfing a residual heterozygous BC1F4 line from a ZM871/ZM895//ZM871 population. A candidate gene TraesCS5B02G044800, encoding an unknown functional protein, was predicted following sequencing and differential expression analyses. There was no significant difference on disease resistance between 5B+ (ZM895 allele) and 5B− (ZM871 allele) homozygous lines based on the powdery mildew and leaf rust reactions. The function of the candidate gene will be verified by an undergoing over-expression experiment in “Fielder” with the ZM871 allele. A KASP marker Kasp-5B-Tgw based on a SNP in the candidate gene was developed and validated in a diverse panel of 166 cultivars. The precise mapping of QTgw.caas-5B laid a foundation for map-based cloning of a predicted causal gene and provided a molecular marker for improvement of grain yield in wheat.