Grain size is a crucial agronomic trait that significantly impacts yield potential in sorghum (Sorghum bicolor), making it a key focus for genetic improvement. In this study, we investigated the genetic basis of grain size variation using two contrasting sorghum accessions, PI302232 (small grain, Sg) and PI563512 (large grain, Lg). The 1000-grain weight, grain length, and grain width of Lg were 3.63-fold, 1.22-fold, and 1.65-fold higher than Sg, respectively. The 1000-grain weight in the F₂ segregating population derived from the cross Sg and Lg parents exhibited the highest phenotypic variation and followed a normal distribution in the three traits. Using bulked segregant analysis sequencing (BSA-seq) with small- and large-grain bulks from the F₂ population, two major quantitative trait loci (QTLs) for grain size were identified on chromosomes 1 and 3. Fine mapping with SSR markers narrowed the qGS1 locus to a 1.03 Mb region on chromosome 1 (Chr01: 22,001,448–23,035,593), containing 49 candidate genes. To narrow down potential candidate genes, transcriptome analysis of spike tissues from Sg and Lg at 0 and 14 days after heading revealed 3719 differentially expressed genes (DEGs), primarily enriched in “starch and sucrose metabolism” and “phenylpropanoid biosynthesis” pathways. Integrating fine mapping intervals and RNA-seq data, 6 DEGs were identified within the qGS1 region. Quantitative real-time PCR confirmed that 6 genes exhibited different expression at two stages. The expression and bioinformatics analysis showed Sobic.001G230700 was the most likely candidate gene for the qGS1 locus. This study provides new insights into the genetic regulation of grain size and a new target to improve grain size in sorghum.