| Summary: | The genome composition of intermediate wheatgrass (IWG) is complex and continues to be a subject of investigation. In this study, molecular cytogenetics were used to investigate the karyotype composition of <i>Th. intermedium</i> and its relative diploid species. St<sub>2</sub>-80 developed from <i>Pseudowroegneria strigose</i> and pDb12H developed from <i>Dasypyrum breviaristatum</i> were used as probes in fluorescence in situ hybridization (FISH) to classify the chromosomes of <i>Th. intermedium</i> into three groups, expressed as <b>J<sup>vs</sup>J<sup>vs</sup>J<sup>r</sup>J<sup>r</sup>StSt</b>. A combined multiplex oligonucleotide probe, including pSc119.2-1, (GAA)<sub>10</sub>, AFA-3, AFA-4, pAs1-1, Pas1-3, pAs1-4, and pAs1-6, was used to establish the FISH karyotype of ten accessions of <i>Th. intermedium</i>. Variability among and within the studied accessions of intermediate wheatgrass was observed in their FISH patterns. Results of this study led to the conclusions that <b>J<sup>vs</sup></b> had largely been contributed from <i>Da. breviaristatum</i>, but not the present-day <i>Da. villosum</i>; IWG had only one <b>J</b> genome, <b>J<sup>r</sup></b>, which was related to either <i>Th. elongatum</i> or <i>Th. bessarabicum</i>; and <b>St</b> was contributed from the genus <i>Pseudoroegneria</i> by hybridization with <i>Th. junceiforme</i> or <i>Th. sartorii</i>.
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