BACKGROUND: This study investigated the molecular mechanisms underlying grain size variation between two distinct naked barley varieties using comprehensive phenotypic and transcriptomic (RNA-Seq) analyses. METHODS: In this study, we employed a comparative transcriptomics approach to analyze two naked barley varieties: the large-grained Shenglibai and the small-grained Lalu Qingke. Our investigation focused on three critical developmental periods of grain growth (early, mid, and late grain-filling periods). By integrating longitudinal three-dimensional phenotypic data with temporal expression profiles and applying weighted gene co-expression network analysis (WGCNA), we successfully identified gene modules that co-vary with morphological expansion. RESULTS: Phenotypic assessments revealed that grains underwent rapid expansion during the filling period, with significant differences in grain width (GW) and thickness (GT) across all three developmental periods. In contrast, grain length (GL) remained relatively consistent by the end of the filling period. Transcriptome sequencing identified a peak in differentially expressed genes (DEGs) during the mid-filling period, indicating that the regulation of grain size development is most active in the early and mid-filling phases. WGCNA identified a blue module strongly correlated with grain size, which was significantly enriched in key metabolic pathways, including starch and sucrose metabolism. Further analysis identified seven hub genes, among which HvENO1 exhibited pronounced upregulation in large-grain varieties during the mid-to-late filling periods, closely aligning with the observed phenotypic traits. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation confirmed the period-specific and variety-specific expression patterns of these genes, further supporting the potential of these genes as targets for improving grain size in breeding.