Many of the catalogued leaf rust resistance genes in wheat deployed in agriculture have been overcome by variants of Puccinia triticina (Pt), the causal pathogen of leaf rust. Discovery and characterization of new sources of resistance in various germplasms using multipathotype tests and molecular markers could permit future diversification of the genetic base of leaf rust resistance in wheat. In searching for new sources of leaf rust resistance, 140 wheat lines from 14 African countries were tested with 8 Australian Pt pathotypes. Seedling tests revealed that 41% of the lines were susceptible to all pathotypes, 31% were postulated to carry either one of 10 resistance genes (Lr1, Lr2a, Lr3a, Lr13, Lr18, Lr23, Lr24, Lr26, Lr37 or Lr73) or one of five gene combinations (Lr2a+Lr3a, Lr1+Lr13, Lr1+Lr23, Lr1+Lr13+Lr73 and Lr23+Lr73). Twenty-eight percent of the lines were postulated to carry uncharacterized seedling resistance genes. Based on average coefficients of infection (ACI), 101, 25 and 11 lines showed high (ACI 0-19), moderate (ACI 21-38) and low (ACI 41-56) levels adult plant resistance, respectively, whereas three lines were moderately susceptible to susceptible (ACI 63-76). Genotyping of 74-78 lines that were anticipated to carry APR genes, using the molecular markers: csLV34 (linked to Lr34) and KASP SNP markers SNP1G22 and SNPT10 (linked to Lr46 and Lr67), respectively, revealed the presence of Lr34, Lr46 and Lr67 in 11, 22 and 14 wheat lines, respectively. The identities of the APR in the remaining 22 lines are unknown, and potentially represent new resistance sources. Genetic analyses of these uncharacterized APR sources are underway to select single gene lines and allow fine mapping.