Five species belonging to the section Sitopsis share the S genome in wild diploid wheat relatives, and the S genome is the B genome origin of durum and bread wheat. Despite of long-term efforts, the donor species of the wheat B genome is still under discussion. Here, we performed RNA sequencing analysis of leaf transcripts derived from the section Sitopsis species to elucidate the phylogenetic relationships between the wheat B genome and the Sitopsis genomes throughout coding regions of each chromosome. Genome-wide polymorphisms including single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were abundantly detected between 19 accessions of the five Sitopsis species and the B genome of common wheat, and the SNPs and indels were efficiently anchored throughout each B genome chromosome. Phylogenetic trees using the nonredundant SNPs showed that the Sitopsis species were clearly divided into two subsections, Truncata and Emarginata, and that the subsection Truncata consisting of Aegilops speltoides Tausch was closer to the wheat B genome than the subsection Emarginata with other four Sitopsis species. However, Ae. Speltoides ssp. speltoides was not well discriminated from Ae. speltoides ssp. ligustica, while the two species, Ae. longissima Schweinf & Muschl. and Ae. sharonensis Eig, were closely related but divided. The fixed nucleotide differences between Ae. longissima and Ae. sharonensis were densely located on the end of the long arm of chromosome 4B, indicating that the distal region of 4SlL might largely control the phenotypic difference of the two species. In addition, most regions on the wheat B genome chromosomes shared the similar topology of the phylogenetic trees based on the entire chromosomes, whereas three regions at the end of chromosomes 3B and 5B showed irregular topology. Genetic divergence between the B genome and Truncata clade was slightly smaller than that between the B genome and Emarginata clade in the distal regions of the chromosomes, which might be related to higher recombination rates in the distal regions of wheat chromosomes and higher proportion of heterozygous in Ae. speltoides by different mating systems between the two subsections: the species of Truncata clade is outcrossing and the species of Emarginata clade is self-pollinating. Namely, viewed from the genome-wide exon sequences, Ae. speltoides is likely to be the direct donor of the entire chromosomes of the wheat B genome.