Wheat (Triticum aestivum L.) is an allohexaploid, in which each of the three genomes has a high 1C content. This indicates the presence of multiple tandemly repeated sequences, which should be detectable using in situ hybridization. Some repeats have already been described, but others remain to be recognized. To discover others, 2000 plasmid wheat clones were examined for signal presence after fluorescence in situ hybridization and microscopic signal observation. Among them, 47 clones produced strong discrete signals on wheat chromosomes. Two of the newly identified clones (pTa-535 and pTa-713) were determined to have especially valuable sequences for chromosome identification. In combination with pTa-86 (the pSc119 homologous sequence), these probes enable unambiguous discrimination of all wheat chromosomes including orientation. Four newly identified sequences (pTa-465, pTa-k566, pTa-s120, and pTa-s126) were useful in that they produced discrete signals on various wheat chromosome arms. Two other clones (pTa-k288 and pTa-k229) produced GISH-like (genomic in situ hybridization) signals because they allowed the A, B, and D genomes to be distinguished simultaneously. In addition, centromere, centromere-related, and ribosomal DNA clones were identified. Also described are improvements on slide preparation and reprobing procedures. To enhance discrete signal detection, a new direct fluorescent-labeling procedure, namely the VentR (exo-) terminal extension method, was employed.