Aegilops umbellulata Zhuk., a wild diploid wheat relative species, has been utilized as genetic resources of several agronomic traits including disease resistance. Nevertheless, the genetic variation has not been comprehensively studied, and the available genome information is still limited. Previously, we performed RNA sequencing of 12 representative accessions of Ae. umbellulata and reconstructed transcripts for each accession (Okada et al. 2018). A large number of genome-wide polymorphisms was obtained and anchored to the genome information of Aegilops tauschii Coss. In this study, we expanded to 112 Ae. umbellulata accessions for evaluation of genetic diversity, and many morphological traits were measured in the Ae. umbellulata accessions. RNA sequencing of the 112 accessions was performed to study the population structure of Ae. umbellulata and to examine the association with the geographic distribution and phenotypic variation. The RNA-seq reads from each accession were aligned to the transcript sequence set of Ae. umbellulata, which was constructed by modifying the reference sequences of transcripts on the D genome of common wheat (IWGSC 2018) with replacement of SNPs between the D genome and Ae. umbellulata based on the RNA-seq reads. More than 2500 SNPs detected among the 112 accessions were distributed throughout the U-genome chromosomes. Based on the SNPs, a phylogenetic tree for the 112 accessions was constructed. The phylogenetic tree and principal component analysis of the polymorphic data showed presence of two major lineages and geographical differentiation in the Ae. umbellulata population. Most accessions belonging to one lineage (lineage 1) were originally collected in Greece, and another lineage (lineage 2) mainly included accessions from other countries such as Turkey, Iran and Iraq. Moreover, lineage 2 was roughly divided to the Turkey subgroup and the Iran/Iraq subgroup. Many phenotypic traits such as heading and flowering time, plant height, awn length and spikelet number were measured in the 112 Ae. umbellulata accessions. Principal component analysis of the phenotypic traits showed significant difference between the subgroups of Turkey and Iran/Iraq populations in the lineage 2. Heading and flowering time, for example, were significantly earlier in the Turkey accessions and later in the Iran/Iraq ones. Flag leaf was significantly longer in the Turkey accessions than in the Iran/Iraq accessions. Taken together, effect of the population structure would be considered to conduct genome-wide association study in Ae. umbellulata. The RNA-seq approach was useful to elucidate the population structure of Ae. umbellulata despite of lacking the reference genome information.