Trigeneric hybrids are commonly used as bridges to transfer genes from some wild species to cultivated wheat and to measure the genomic interaction between donor species. We previously reported that trigeneric germplasms were produced by crossing wheat-Psathyrostachys huashanica amphiploids (PHW-SA, 2n = 8x = 56, AABBDDNsNs) with hexaploid triticale (Zhongsi 828, 2n = 6x = 42, AABBRR). In the present study, chromosome pairing behavior and the genome constitution of the F4 progenies of wheat-rye-P. huashanica trigeneric hybrids were studied. Cytological analysis showed that the chromosome number of F4 progenies ranged from 39 to 46, and 57.5% of them had 42 chromosomes. The mean meiotic configuration of F4 lines was 1.71 univalents, 20.26 bivalents, 0.04 trivalents, and 0.001 quadrivalents per pollen mother cell. Among the lines with 2n = 42, the average pairing configuration was 1.21 univalents, 16.22 ring bivalents, 4.16 rod bivalents, and 0.01 trivalents. This result indicated that these lines were cytologically stable. Other lines with 2n = 39, 40, 41, 43, 44, 45, and 46, bearing a high number of univalents or multivalents, showed abnormal meiotic behavior. Genomic in situ hybridization (GISH) revealed that all F4 lines had 11-14 rye chromosomes, but no P. huashanica chromosomes. The complete set of 14 rye chromosomes was found in 19 lines. At meiosis, GISH detected 1-6 univalents with hybridization signals of rye in 13 lines. Bivalents with fluorescence signals were identified in each line, ranging from 3 to 7. A quadrivalent with hybridization signals was observed in only 1 line, K13-714-8. Lagging chromosomes, chromosome bridges, micronuclei, and chromosome fragments hybridizing with the probe were not discovered in any of the lines. These results inferred that the behavior of rye chromosomes was normal during meiosis. In addition, 21 lines of 2n = 42 (91.3%) with 12 or 14 rye chromosomes, always contained 6 or 7 bivalents bearing fluorescence signals. This suggested that the rye chromosomes exhibiting complete pairing in these lines were cytologically stable during meiosis and may therefore be considered as new hexaploid triticales. Thus, these lines might be potential materials for further hexaploid triticale improvement.