The rise in human population necessitates the use of all available tools to enhance wheat productivity. In this regard, pre-breeding has mobilized novel under-utilized genetic variation into breeding programs. However, this germplasm needs to be characterized for its efficient utlization. This investigation was initiated to evaluate the early and late sown wheat pre-breeding germplasm for physiology and yield related traits and to associate the mapped SNPs using association mapping approach. Our results indicate that the germplasm performed better in early sowing in comparison to late planting where grain yield (Yd) was found positively correlated with water use efficiency (WUE), heading time and chlorophyll contents (Chl). We discovered a total of 210 associations involving 155 SNPs. Taking into consideration either early or late sowing and the mean values only 12 markers traits were involved with germination, plant height, stomatal conductance, transpiration rate, Chl, carotenoids and Yd. Our correlations and mapping results indicate that higher WUE along-with Chl can be targeted as indirect physiological markers to enhance wheat yield.