Effective at the adult plant stage, Lr34 is the most important resistance gene to leaf rust. Usage of closely linked molecular markers is the best strategy to facilitate the incorporation of economically important genes in an adapted plant germplasm. Ten novel molecular markers spanning the Lr34 locus were developed, including six microsatellites (cam), one insertion site-based polymorphism (caISBP), two single nucleotide polymorphisms (caSNP) and one indel marker (caIND). Marker caIND11 is the best diagnostic marker for marker assisted selection of Lr34. Two novel haplotypes of Lr34 were discovered in the germplasm. Analysis of these markers on five segregating populations revealed a recombination between caSNP4 and cam8 which provided further support for the identity of the ABC transporter as Lr34. Using Lr34-specific markers, the world collection (WC) was divided into five major haplotypes (H) of which H1 was consistently associated with the resistance phenotype Lr34+. SNP12-C is the functional unit of Lr34. Maximum parsimony network and other observations revealed that H4, an Lr34- haplotype, was probably the most ancient haplotype and H1 the most recent and that it likely arose after the advent of hexaploid wheat. Analysis of geographical distribution showed that H1 was at a high frequency in the Asian germplasm while H4 was more frequent in the European germplasm. Lr34, a gain of function mutation, was hypothesized to have originated in Asia. The (WC) was characterized for seedling and adult plant resistance using gene specific markers and gene postulation. Fourteen seedling genes were determined or postulated in the collection. Lr1, Lr10, Lr3 and Lr20 were the most highly represented genes while Lr9, Lr14b, Lr3ka and/or Lr30 and Lr26 were rare. The WC was evaluated for field resistance. The rust rating in the field ranged from nearly immune (1R) to highly susceptible (84S). Most Lr34 containing accessions had maximum rust severity (MRS) of 35%. The high levels of resistance in some accessions are likely the result of synergy between APR genes or between APR and seedling genes. Accessions that were highly resistant should be considered potential sources of resistance for future wheat breeding program to improve leaf rust resistance.