Wheat yellow rust epidemics, caused by Puccinia striiformis f. sp. tritici (PST) have increased worldwide with the emergence in Western Europe and North Africa of an aggressive strain and tolerant to high temperature (PstS2) since 2004 and exotic strains (Triticale 2006, 2015, Warrior and Kranich) recently. Even though resistant varieties bring to agriculture effective solutions to reduce the use of pesticides, races of the pathogen quickly overcome introduced resistance genes. In this study we determined the virulence combinations using the European and world differential sets and the simple sequence repeat diversity of 94 PST isolates collected in Tunisia since 2013 from the major wheat growing areas. In addition, 39 local Tunisian durum wheat accessions and 109 recombinant inbred lines RILs (F9) were evaluated at the seedling stage against old and new races of PST in order to identify resistant genotypes and markers associated with resistance genes. The results underlined the rapid change in wheat yellow rust population where 3 predominant races, Warrior1 (W1), Warrior- (W-) and Triticale 2006 were detected since 2013. In addition, 10% of the durum wheat accessions were resistant to W1, W- and PstS2. The evaluation of the RILs issued from a cross between Tunisian durum wheat Khiar and Agili39 revealed one major resistance gene against W1 and two minor genes against PstS2. The use of these genes in breeding can improve durability of the genetic control against PST