| Summary: | <i>Thinopyrum</i> has been widely used to improve wheat (<i>Triticum aestivum</i> L.) cultivars. Non-denaturing fluorescence in situ hybridization (ND-FISH) technology using oligonucleotides (oligo) as probes provides a convenient and efficient way to identify alien chromosomes in wheat backgrounds. However, suitable ND-FISH-positive oligo probes for distinguishing <i>Thinopyrum</i> chromosomes from wheat are lacking. Two oligo probes, Oligo-B11 and Oligo-pThp3.93, were designed according to the published <i>Thinopyrum ponticum</i> (<i>Th. ponticum</i>)-specific repetitive sequences. Both Oligo-B11 and Oligo-pThp3.93 can be used for ND-FISH analysis and can replace conventional GISH and FISH to discriminate some chromosomes of <i>Th. elongatum</i>, <i>Th. intermedium</i>, and <i>Th. ponticum</i> in wheat backgrounds. The two oligo probes provide a convenient way for the utilization of <i>Thinopyrum</i> germplasms in future wheat breeding programs.
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