To preliminarily evaluate the genetic diversity of the Iranian diploid Triticum L. gene pool, in this study, a total of 176 individuals belonging to T. monococcum L. ssp. aegilopoides (Link) Thell. and T. urartu Thum. ex Gandil. were pre-screened using single-strand conformation polymorphism (SSCP) analysis of the Acc-1 and Pgk-1 loci. A selected set of 76 DNA samples corresponding to the observed SSCP variants were sequenced for both loci and evaluated for nucleotide diversity associated with the taxonomic groups and geographical regions. We found three haplotypes, including one that was new for Iran, at each locus. Population structure and analysis of molecular variation results proved that the collection evaluated could be genetically divided into two distinct groups, which to a great extent was in accordance with the taxonomic classification. A genetic leakage from T. monococcum ssp. aegilopoides into T. urartu was observed during structure analysis, which was inferred on the basis of occasional outcrossing, despite their inbreeding nature. The results revealed that there was no variation within the populations belonging to T. urartu, while a meaningful variation was found between the geographical regions for T. monococcum ssp. aegilopoides. The median-joining networks revealed a conflict between morphology and haplotype variation, which was interpreted on the basis of introgressive hybridization, recombination signature and rapid speciation. In conclusion, we suggest that SSCP analysis is a useful tool in regions where thorough sequencing of an enormous number of DNA samples is time consuming and not affordable.