Root architecture is intricately linked to the acquisition of water and nutrients in maize seedlings. Despite its functional importance, few genes controlling root development have been targeted for drought resistance in breeding. Here, we performed a genome-wide association analysis to detect genetic variants linked to primary root length (PRL) across 307 inbred lines grown under hydroponic conditions. We identified 28 SNPs significantly associated with 25 candidate genes, accounting for 6.09%–11.07% of the phenotypic variation. Among them, ZmHSP20-5, encoding a cytoplasm-localized small heat shock protein (sHSP) with preferential expression particularly in lateral root primordia emerged as a promising candidate. Functional validation using knockout mutants revealed that disruption of ZmHSP20-5 impaired root architecture, causing reduced primary root elongation, shorter lateral roots, decreased lateral root density, and compromised drought tolerance. Further analysis revealed that InDel-1224 in the ZmHSP20-5 promoter likely contributed to differential gene expression and variation in root development among inbred lines. Evolutionary evidence suggested that the ZmHSP20-5 locus may have undergone selection during domestication, with the favorable ZmHSP20-5^In-1224 allele increasing in frequency over time. Overall, these findings establish that natural variation in ZmHSP20-5, particularly the ZmHSP20-5^In-1224 allele, contributes to root growth and drought resistance, providing a valuable genetic resource for the breeding of drought-resistant maize varieties with optimized root systems.