To transfer multiple desirable alien genes into common wheat, we previously reported a new trigeneric hybrid synthesized by crossing a wheat - Thinopyrum intermedium partial amphiploid with wheat - Psathyrostachys huashanica amphiploid. Here, the meiotic behavior, chromosome constitution, and stripe rust resistance of F5 derivatives from the wheat - Th. intermedium - P. huashanica trigeneric hybrid were studied. Cytological analysis indicated the F5 progenies had chromosome numbers of 42-50 (average 44.96). The mean meiotic configuration was 1.28 univalents, 21.74 bivalents, 0.04 trivalents, and 0.02 tetravalents per pollen mother cell. In 2n = 42 lines, the average pairing configuration was 0.05 I + 19.91 II (ring) + 1.06 II (rod) + 0.003 IV, suggesting these lines were cytologically stable. Most lines with 2n = 43, 44, 46, 48, or 50, bearing a high frequency of univalents or multivalents, showed abnormal meiotic behavior. Genomic in situ hybridization karyotyping results revealed that 25 lines contained 1-7 Th. intermedium chromosomes, but no P. huashanica chromosomes were found among the 27 self-pollinated progenies. At meiosis, univalents (1-5) possessing Th. intermedium hybridization signals were detected in 19 lines. Bivalents (1-3) expressing fluorescence signals were observed in 12 lines. Importantly, 21 lines harbored wheat - Th. intermedium chromosomal translocations with various alien translocation types. Additionally, two homozygous lines, K13-668-10 and K13-682-12, possessed a pair of wheat - Th. intermedium small fragmental translocations. Compared with the recurrent parent Zhong 3, most lines showed high resistance to the stripe rust (Puccinia striiformis f. sp. tritici) pathogens prevalent in China, including race V26/Gui22. This paper reports a highly efficient technical method for inducing alien translocation between wheat and Th. intermedium by trigeneric hybridization. These lines might be potentially valuable germplasm resources for further wheat improvement.