Triticale (×Triticosecale), an amphiploid of wheat (mainly Triticum turgidum) and cereal rye (Secale cereale), is an excellent source of resistance to wheat stem rust, caused by Puccinia graminis f. sp. tritici. A collection of 567 triticale accessions originating from 21 countries was evaluated at the seedling stage for reaction to races of P. graminis f. sp. tritici with broad virulence, including TTKSK, TRTTF, and TTTTF. A high frequency (78.4%) of accessions was resistant to race TTKSK, with low infection types ranging from 0; to X. A selection of 353 TTKSK-resistant accessions was evaluated for reaction to three South African isolates of P. graminis f. sp. tritici with single and/or combined virulences to stem rust resistance genes SrSatu, Sr27, and SrKw present in triticale. Genes SrSatu, Sr27, and SrKw were postulated to be present in 141 accessions and contributed to TTKSK resistance. The remaining 212 resistant accessions may possess uncharacterized genes or combinations of known genes that could not be determined with these isolates. These accessions were further evaluated for resistance to races TTKST, TPMKC, RKQQC, RCRSC, QTHJC, QCCSM, and MCCFC. Resistance remained effective across the entire set of races in the majority of the accessions (n = 200), suggesting that the resistances are effective against a broad spectrum of virulence. In all, 129 (79.6%) resistant accessions with noncharacterized genes were resistant to moderately resistant in field stem rust nurseries at Debre Zeit (Ethiopia) and St. Paul (Minnesota). Results from evaluating F2 populations derived from resistant-susceptible crosses revealed that resistance to TTKSK in triticale was conferred mostly by single genes with dominant effects.