In cereal crops such as barley (Hordeum vulgare L.), pre-anthesis tip degeneration (PTD) starts with growth arrest of the inflorescence meristem dome, followed basipetally by the degeneration of floral primordia and the central axis. Due to its quantitative nature and environmental sensitivity, inflorescence PTD constitutes a complex, multilayered trait affecting final grain number. This trait was studied by microscopic dissection of immature inflorescence meristems under standardized growth conditions. We combined spatiotemporal metabolomic, transcriptomic, and genetic approaches to elucidate the mechanism of barley inflorescence PTD in two- and six-rowed barley cultivars ‘Bowman’ and ‘Morex,’ respectively. Metabolome profiling includes hormones and primary metabolites such as sugars, TCA intermediates, and amino acids by dividing spike meristems into dying apical and viable central and basal parts at four developmental stages during the spike growth phase. Similarly, RNA sequencing was performed for three developmental stages in both genotypes. RNA sequencing data analyses were performed to identify differentially expressed and tissue-specific transcripts. Further, PTD-associated hub genes were identified by weighted gene coexpression network analysis. Based on transcriptome analyses, we identified an important modulator of inflorescence PTD and functionally validated it using Cas9-mediated mutagenesis and gene-based associated study using a diverse panel of barley accessions.