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Four Ug99 pathotypes occur in southern Africa. Although South African bread wheat cultivars and lines are regularly screened against representative isolates, the stem rust reactions of Zimbabwean germplasm to these variants were largely unknown. A collection of 49 wheat cultivars and lines, obtained from Seed-Co (Ltd.) and the Crop Breeding Institute in Zimbabwe, were tested as seedlings against pathotypes TTKSF, TTKSF+, TTKSP and PTKST. Twelve varieties and 21 experimental lines showed low infection types with all four pathotypes. Using molecular marker assays Sr31 was detected in 26 entries, Sr24 in five and Sr36 in one. The csSr2 marker suggested the presence of Sr2 in 20 entries. Screening of adult plants in the greenhouse using pathotype PTKST showed 34 entries with low infection types and 15 had high infection types. Stem rust field records in 2012 showed 5 susceptible entries with stem rust scores between 50S and 80S, whereas only 4 susceptible entries were identified in 2014 with scores ranging from 30S to 80S. Three lines were susceptible in both seasons. The study exposed the vulnerability of Zimbabwean wheat germplasm to Ug99 variants, but also identified suitable lines that can be used in breeding and possible commercialization.