Domesticated timopheevi wheat (Triticum timopheevi) is an endemic crop of western Georgia in Transcaucasia. It has a distinct nuclear genome (2n = 28, AAGG) and is genetically isolated from other wheat species. To clarify the genetic diversity and the domestication of this interesting wheat, we analyzed molecular variation at 23 microsatellite loci in the chloroplast genome. Allelic diversity was evaluated using 94 accessions representing domesticated timopheevi wheat (T. timopheevi), wild timopheevi wheat (T. araraticum), and wild emmer wheat (T. dicoccoides). The average diversity index (H) in T. araraticum (0.206) was smaller than that in T. dicoccoides (0.284). No polymorphisms were detected among the six accessions of T. timopheevi, suggested a monophyletic origin of domesticated timopheevi wheat. Phylogenetic analyses of the plastotypes revealed clear differences between the chloroplast DNA of timopheevi wheat and emmer wheat, and thus supported the hypothesis that these two wheat species originated independently. None of the T. araraticum plastotypes collected in Transcaucasia were closely related to the T. timopheevi plastotype. On the other hand, the plastotypes found in northern Syria and southern Turkey showed closer relationships with T. timopheevi. These results suggested that the domestication of timopheevi wheat might have occurred in the region including southern Turkey and northern Syria.