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Preventing the widespread occurrence of stripe rust in wheat largely depends on the identification of new stripe rust resistance genes and the breeding of cultivars with durable resistance. In previous study, we reported 6E of wheat-tetraploid Thinopyrum elongatum 6E (6D) substitution line contains adult-stage stripe rust resistance genes. In this study, three novel wheat-tetraploid Th. elongatum translocation lines were generated from the offspring of a cross between common wheat and the 6E (6D) substitution line. Genomic in situ hybridization (GISH), fluorescence in situ hybridization chromosome painting (FISH painting), repetitive sequential FISH, and 55 K SNP analyses indicated that K227-48, K242-82, and K246-6 contained 42 chromosomes and were 6DL·6ES, 2DL·6EL, and 6DS·6EL translocation lines, respectively. The assessment of stripe rust resistance revealed that K227-48 was susceptible to a mixture of Pst races, whereas the 6EL lines K242-82 and K246-6 were highly resistance to stripe rust at the adult stage. Thus, this resistance was due to the chromosome arm 6EL of tetraploid Th. elongatum . The improved agronomic performance of 6DS·6EL translocation line may be a useful novel germplasm resource for wheat breeding programs. For the application of marker-assisted selection (MAS), 47 simple sequence repeat (SSR) markers were developed, showing specific amplification on the chromosome 6E using the whole-genome sequence of diploid Th. elongatum . The 6DS·6EL translocation line and SSR markers have the potential to be deploy for future stripe rust resistance wheat breeding program., Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01493-6., Competing Interests: Competing InterestsThe authors declare that they have no conflict of interest., (© The Author(s), under exclusive licence to Springer Nature B.V. 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)