Triticum boeoticum (2n = 2x = 14, AbAb) contains beneficial traits for common wheat improvement. The discrimination of Ab-genome chromosomes from A-genome chromosomes is an important step in gene transfer from T. boeoticum to common wheat. In this study, fluorescence in situ hybridization (FISH) analysis using nine oligonucleotide probes revealed high divergence between chromosomes of the common wheat germplasm Crocus and T. boeoticum accession G52. The combination of Oligo-pTa535-HM and Oligo-pSc119.2-HM can differentiate Ab and A chromosomes within homologous groups 2, 4, 5, and 6; chromosomes 2Ab and 6Ab can be identified by using (ACT)7, (CTT)7, and (GAA)7. The probes Oligo-pTa713 and (ACT)7 can be utilized for the identification of chromosomes 1Ab and 3Ab, respectively. Probes (CAG)7 and (CAC)7 can be applied in the identification of 7Ab. Moreover, probe combinations consisting of Oligo-pTa535-HM and (AAC)7 with (ACT)7 or (CTT)7 and of Oligo-pTa535-HM and Oligo-pTa713 with (CAC)7 or (CTT)7 will help discriminate the Ab-genome chromosomes of T. boeoticum. These probes are being used as potential markers to select common wheat Crocus-T. boeoticum G52 alien chromosome lines. Moreover, FISH patterns are highly divergent between Ab- and A-genome chromosomes, indicating that obvious chromosome structural variations arose during wheat evolution.