The Sando collection, consisting of over 490 accessions derived from interspecific and intergeneric hybridization between Triticum and related species, was developed by the late W. J. Sando between 1930 and 1940 and is currently maintained at the USDA National Small Grains Germplasm Research Facility in Aberdeen, ID. This study is the first comprehensive cytological and seed morphological investigation of the collection. Cytological methods, including genomic in situ hybridization (GISH), have determined 2n chromosome numbers and identified chromosomes from Triticum and other species in 250 accessions. Digital imaging software captured variations in seed color, size and shape. A chromosome count of 2n=42 was found in the majority of the accessions (204) analyzed to date. The remaining accessions exhibited ranges of 2n chromosome numbers including 42 to 56; 40 to 44 with fragments; 44 to 56; 52 to 56; 28; 28 to 42; 28 to 56 and 21 to 42. Agropyron (Thinopyrum) chromosomes were identified by GISH in several accessions with 2n>42. These materials are valuable germplasm sources for breeding for disease-resistance and forage production.