Grain yield establishment is a complex progress and the genetic basis of one of the most important yield components, 100-kernel weight, remains largely unknown. Here, we employed a double haploid (DH) population containing 477 lines which was developed from a cross of two maize elite inbred lines, PHBA6 and Chang7-2, to identify quantitative trait loci (QTL) that related to 100-kernel weight. The phenotypes of the DH population were acquired over three years in two different locations, while the DH lines were genotyped by next-generation sequencing technology of massively parallel 3ʹ end RNA sequencing (MP3RNA-seq). Eventually, 28,874 SNPs from 436 DH lines were preserved after SNP calling and filtering and a genetic map with a length of 837 cM was constructed. Then, single environment QTL analysis was performed using the R/qtl program, and it was found that a total of 17 QTLs related to 100-kernel weight were identified and distributed across the whole genome except chromosomes 5 and 6. The total phenotypic variation explained by QTLs detected in three different environments (BJ2016, BJ2107, and HN2018) was 22.2%, 32.9%, and 51.38%, respectively. Among these QTLs, three of them were identified across different environments as environmentally stable QTLs and explained more than 10% of the phenotypic variance each. Together, the results provided in this study preliminarily revealed the genetic basis of 100-kernel weight and will enhance molecular breeding for key agronomic kernel-related traits in maize.