A survey of genome-wide polymorphisms between closely related species is required to understand the molecular basis of the evolutionary differentiation of their genomes. Two wild diploid wheat species, namely Triticum monococcum ssp. aegilopoides and T. urartu, are closely related and harbour the Am and A genomes, respectively. The A-genome donor of tetraploid and common wheat is T. urartu, and T. monococcum ssp. monococcum is the cultivated form derived from the wild einkorn wheat subspecies aegilopoides. Although subspecies aegilopoides has been a useful genetic resource in wheat breeding, genome-wide molecular markers for this subspecies have not been sufficiently developed. Here, we describe the detection of genome-wide polymorphisms such as single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) from RNA sequencing (RNA-seq) data of leaf transcripts in 15 accessions of the two diploid wheat species. The SNPs and indels, detected using the A genome of common wheat as the reference genome, covered the entire chromosomes of these species. The polymorphism information facilitated a comparison of the genetic diversity of einkorn wheat with that of two related diploid Aegilops species, namely, Ae. tauschii and Ae. umbellulata. Cleaved amplified polymorphic sequence (CAPS) markers converted from the SNP data were efficiently developed to confirm the addition of aegilopoides subspecies chromosomes to tetraploid wheat in nascent allohexaploid lines with AABBAmAm genomes. In addition, the CAPS markers permitted linkage map construction in mapping populations of aegilopoides subspecies accessions. Therefore, these RNA-seq data provide information for further breeding of closely related species with no reference genome sequence data.