Grain number and size determine grain yield in crops and are closely associated with spikelet fertility and grain filling in barley (Hordeum vulgare). Abortion of spikelet primordia within individual barley spikes causes a 30%–50% loss in the potential number of grains during development from the awn primordium stage to the tipping stage, after that grain filling is the primary factor regulating grain size. To identify transcriptional signatures associated with spike development, we use a six-rowed barley cultivar (Morex) to develop a spatiotemporal transcriptome atlas containing 255 samples covering 17 stages and 5 positions along the spike. We identify several fundamental regulatory networks, in addition to key regulators of spike development and morphology. Specifically, we show HvGELP96, encoding a GDSL domain-containing protein, as a regulator of spikelet fertility and grain number. Our transcriptional atlas offers a powerful resource to answer fundamental questions in spikelet development and degeneration in barley.