Leaf rust, caused by Puccinia triticina (Pt), is an important disease of wheat in Pakistan and many other wheat growing countries. Seventy six leaf rust resistance genes have been formally named based on genetic analysis in different mapping populations. A majority of these genes belong to the all stage resistance (ASR) category. Only a small proportion of these genes are currently effective against Pt pathotypes of Pakistan. Genetic analysis based on a large set of bi-parental crosses can be time-consuming, expensive and technically demanding. In contrast, association analysis using phenotypic and phenotype-neutral genotypic data can be used to understand existing genetic diversity for a given trait and to uncover the uncharacterized loci. Four hundred (400) wheat genotypes collected from different institutes of Pakistan, CIMMYT, ICARDA and other countries were evaluated for leaf rust resistance under artificial inoculation by using mixture of inoculum during two consecutive years. A set of 94 wheat genotypes representing the available genetic diversity were then shortlisted for detailed genetic characterization and was genotyped using 203 microsatellite (SSR) markers covering all 21 wheat chromosomes. Marker-leaf rust associations were determined using single marker tests. Markers located on chromosomes 1DS, 4AL, 4BL, 5AS, 5BS, 6AL, 6BS and 6DS showed significant associations with leaf rust response variation. These genotypes were also scored for variation in days to heading, days to maturity, plant height, number of grains per spike, yield per plant, 1000 grain weight, biological yield and harvest index. Significant marker-trait associations were observed for all these traits.