Stripe rust, a serious threat to food security, has a worldwide distribution and also responsible for up to 70% yield losses in wheat. There is a dire need to develop stripe rust resistant varieties which is an environment friendly and ultimate solution to minimize yield losses in wheat. The present study was aimed to investigate the genetic diversity in a segregating population’s genome that is enriched with yellow rust genes and their interactions with agro-morphological traits. In this study, two segregating wheat populations (NIGAB-08 and NIGAB-09), which were developed at National Institute of Genomics and Advanced Biotechnology (NIGAB) have been analyzed at seedling and adult plant stage against stripe rust along with its effects on yield contributing traits. Further, it was screened for the presence of stripe rust resistance genes by using various available molecular markers. In all, NIGAB-08 (F5 and F6) segregating population comprised of 50 wheat lines and NIGAB-09 (F5 and F6) segregating populations having 86 wheat lines have been evaluated for yield contributing traits in the consecutive two years. Among them, 50 wheat lines of NIGAB-08 and 60 wheat lines of NIGAB-09 were evaluated for genetic variation in markers linked with stripe rust in consecutive two years. Results have shown that Coefficient of Infection (CI) value has a negative correlation with yield per plant by direct or indirect effects on other traits including tillers per plant, spikelet per spike, spike length and 1000 grains weight. Moreover, it has been observed that spike length, spikelet per spike and thousand grain weight have a positive correlation with yield per plant. Regression analysis in NIGAB-08 showed the same results that spikelet per spike, spike length and 1000 grains weight are the significant yield contributing traits. Then the segregating wheat populations were evaluated for yellow rust resistance at seedling and adult plant stage. In NIGAB- 08, 87% of the population was resistant, 2% having the intermediate type of resistance and 10% was susceptible at seedling stage (in greenhouse experiment) that indicate the presence of major yellow rust (Yr) resistant genes in this population. While NIGAB-09 wheat population was observed for 83% resistant, 5% intermediate and 13% of the population found as susceptible. It showed that the population is enriched with major Yr genes. NIGAB-08 showed 16% of the population was resistant, 47% showed an intermediate type of resistance while 35% was found as susceptible at adult plant stage. These results indicate that NIGAB-08 wheat population has less number of minor Yr genes as compared to major Yr genes. In Field evaluation, 79% of NIGAB-09 population was scored as resistant, 11% with intermediate type of resistance and 10% as susceptible. These results showed that the population is also enriched with minor Yr genes along with some major genes. Both wheat segregating populations (NIGAB-08 and NIGAB-09) were assayed for the presence of stripe rust resistance genes by using 27 SSR and microsatellite markers. In NIGAB-09 wheat population, on the basis of distance of marker from target gene, Yr5 showed 56% presence in F5 population and 55% in F6 whereas; Yr7, Yr15, Yr17, Yr18, Yr26, Yr29, Yr35 Yr36, YrCN19/Yr41 and YrHua showed 58%, 70%, 70%, 45%, 70%, 52%, 64%, 78%, 26% and 56% presence in first year of study respectively ( F5 Population) and 70%, 62%, 92%, 47%, 90%, 70%, 70%, 70%, 75% and 83% in second year of study (F6 population), respectively. In NIGAB-08 segregating wheat population, it had been observed that frequency of Yr5 was 84% in F5 segregating lines and 88% in F6 population while the Yr7 genes was 44% present in F5 and 30% in F6 population whereas Yr15, Yr17, Yr18, Yr26, Yr29, Yr35 Yr36, YrCN19/Yr41 and YrHua were 72%, 54%, 68%, 56%, 94%, 46%, 56%, 54% and 48% abundant in the first year of study (F5 Population) while, 94%, 46%, 88%, 70%, 92%, 48%, 62%, 50% and 62% in consecutive next year of study (F6 segregating population). Dendrograms have been developed for both populations on the basis of low Coefficient of Infection value, higher Thousand Grain Weight and Yield per plant (values) to identify the slow rusting lines in these segregating wheat populations along with best agro-morphological traits. One line of NIGAB-08 (Line no. 40) and eighteen lines of NIGAB-09 (Line no. 2, 6, 10, 17, 20, 21, 22, 27, 30, 35, 39, 49, 82, 84, 89, 94, 97, and 108) have shown best results in terms of seedling screening, field evaluation against stripe rust along with agro-morphological traits and in molecular evaluation in these wheat lines. When wheat lines were selected on the basis of best results at seedling stage and adult plant resistance, the two lines of NIGAB-08 (23, 36) and 35 wheat lines of NIGAB-09 (5, 16, 19, 24, 26, 28,29, 31, 32, 33, 34, 36, 37, 40, 42, 44, 45, 46, 52, 54, 57, 60,61, 62, 68, 70, 71, 73, 83, 86, 95, 98,105, 109, 110) were found in this category. Nine wheat lines of NIGAB-08 (4, 11, 10, 35, 63, 41, 32, 53, 61) and twenty wheat lines of NIGAB-09 (51, 8, 69,107, 3, 88, 85, 65, 93, 87, 27, 92, 76, 74, 12, 75,106, 13, 25, 38) have shown good results in screening at adult plat stage with best agro-morphological traits and in molecular evaluation. This study has been focused on to screen segregating wheat populations in all aspects, which are enriched with stripe rust resistance genes which can further be utilized in developing commercial wheat varieties. Some novel genes have also been identified during these studies which were not previously reported in Pakistani wheat cultivars.