The 100-kernel weight of maize is a main component of yield traits. The research of genetic controlling sites by identifying QTL or cloning genes is helpful for the implementation of molecular breeding. In this study, We used introgressed homozygous line SL19-41, containing two chromosome fragments of QB80 introgressed into the genetic backgrounds of Ye478, because Ye478 is a main maize inbred line with widely used in maize breeding. We constructed the segregated population families by hybridizinng with Ye478 (F2、F2:3 and BC1F1). After the field experiment of three environments, we located the 100-kernel weight QTL and analyzed the phenotypic effects of QTL loci linked markers through the stepwise regression interval mapping by IciMapping software. The results showed that two QTLs of 100-kernel weight were identified, of which qKW4-1 located in bnlg1784-umc1194 interval on the 4 chromosome which are repeatly detected in three environments, interpreting 6.74%-17.81% phenotypic variations and illustrated genetic mechanisms of 100-kernel weight of introgression line SL19-41, At the same time, we obtained the modified version of the Ye478 (Ye478QB80), it provided useful molecular markers for genetic improvement of 100-kernel weight of maize and also provided material resources for cloning genes of 100-kernel weight.