Wild diploid wheat Aegilops tauschii, the D-genome progenitor of common wheat, carries large genetic variation in spikelet and grain morphology. Two differentiated subspecies of Ae. tauschii, subspecies tauschii and strangulata, have been traditionally defined based on differences in spikelet morphology. Here, we first assessed six spikelet shape-related traits among 199 Ae. tauschii accessions, and found that the accessions belonging to TauL1major lineage produced significantly longer spikes, higher spikelet density, and shorter, narrower spikelets than another major lineage, TauL2, in which the strangulata accessions are included. Next, we performed quantitative trait locus (QTL) analysis of the spikelet and grain shape using three mapping populations derived from interlineage crosses between TauL1 and TauL2 to identify the genetic loci for the morphological variations of the spikelet and grain shape in Ae. tauschii. Three major QTL regions for the examined traits were detected on chromosomes 3D, 4D and 7D. The 3D and 4D QTL regions for several spikelet shape-related traits were conserved in the three mapping populations, which indicated that the 3D and 4D QTLs contribute to divergence of the two major lineages. The 7D QTLs were found only in a mapping population from a cross of the two subspecies, suggesting that these 7D QTLs may be closely related to subspecies differentiation in Ae. tauschii. Thus, QTL analysis for spikelet and grain morphology may provide useful information to elucidate the evolutionary processes of intraspecific differentiation.