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Abstract Background Partial amphiploids created by crossing octoploid tritelytrigia(2n = 8× = 56, AABBDDEE) and Thinopyrum intermedium (2n = 6× = 42, StStJJJSJS) are important intermediates in wheat breeding because of their resistance to major wheat diseases. We examined the chromosome compositions of five wheat-Th. intermedium partial amphiploids using GISH and multicolor-FISH. Results The result revealed that five lines had 10-14 J-genome chromosomes from Th. intermedium and 42 common wheat chromosomes, using the J-genomic DNA from Th. bessarabicum as GISH probe and the oligo probes pAs1-1, pAs1-3, AFA-4, (GAA) 10, and pSc119.2-1 as FISH probe. Five lines resembled their parent octoploid tritelytrigia (2n = 8× = 56, AABBDDEE) but had higher protein contents. Protein contents of two lines HS2-2 and HS2-5 were up to more than 20%. Evaluation of Fusarium head blight (FHB) resistance revealed that the percent of symptomatic spikelets (PSS) of these lines were below 30%. Lines HS2-2, HS2-4, HS2-5, and HS2-16 were less than 20% of PPS. Line HS2-5 with 14 J-genome chromosomes from Th. intermedium showed the best disease resistance, with PSS values of 10.8% and 16.6% in 2016 and 2017, respectively. Conclusions New wheat-Th. intermedium amphiploids with the J-genome chromosomes were identified and can be considered as a valuable source of FHB resistance in wheat breeding.