Old germplasm is an important genetic resource for enhancing modern crops with new alleles. In the present study, the genetic diversity of 52 historic varieties and landraces of bread wheat originated from the Western (Bulgaria) and Northeastern (Ukraine, Russia and Georgia) regions of the Black Sea basin was assessed based on microsatellite markers and agronomic characteristics. A set of 24 markers detected a total of 263 alleles at 25 microsatellite loci, with an average number of 10.5 alleles per locus and an average polymorphic information content (PIC) of 0.74. A total of 63 alleles at 22 loci were unique, being specific to a particular accession. Half of the alleles (132) were regionally specific, and the rest were common between the Western and Northeastern accessions. The latter group was characterized with greater total and private allelic richness, a higher number of unique alleles and a higher average PIC. The population was found to be very heterogeneous (average heterogeneity 41%), with the Northeastern pool (52.8%) being more diverse than the Western pool (30.9%). Most of the accessions of the Western group clustered together, and the rest were distributed among the subclusters of the Northeastern germplasm. Large inter-group differences in the frequencies of alleles ranging from 3.1 at Xgwm294-2A to 16.7 at Xgwm333-7B were observed. This variation might partly account for the differences in certain yield-related traits. The Northeastern accessions had significantly longer spikes with more number of spikelets. Some issues related to germplasm preservation in seed genebanks are discussed herein. The large molecular variation observed could be utilized by breeders for the selection of diverse parents, or by researchers for the production of mapping populations.