Yield, of which kernel weight is a major component, is the primary trait of interest in maize breeding programmes. In our previous study, a major QTL (named qhkw5-3), which controls hundred-kernel weight, was identified and mapped to the interval between simple sequence repeat (SSR) markers SYM033 and SYM108 on chromosome 5, using an F2:3 population derived from a cross between the maize inbred line Zheng58 and the single-segment substitution line Z22. In order to fine map qhkw5-3, a larger BC1F1 segregating population of 14,759 seeds, derived from a (Z22 × Zheng58) × Z22 cross, was screened using the SSR markers SYM036 and SYM119. Forty genotypes with donor chromosomal fragments of different lengths were obtained. Progeny testing results indicated that qhkw5-3 was mapped to an interval of 442.6 kb between the SSR markers SYM077 and SYM084. Overlap mapping results, based on seven homozygous recombinant lines, showed that qhkw5-3 was narrowed down to an interval of 125.3 kb between the InDel markers InYM20 and InYM36. Within this interval, six candidate genes were analysed using qRT-PCR. The results of this study lay the foundations for cloning and functional analysis of qhkw5-3 and will contribute to advancing our knowledge of the genetic basis of yield traits in maize.