The field study was conducted to evaluate the effect of tillage, crop residue retention and crop rotationon proline concentration in flag leaves of wheat as an important factor influencing future grain quality and yield during the grain filling period. Conservation agriculture (CA) is widely adopted by the farmers but due to remaining uncertainties about its effects on quality crop yield, needs to be explained through study of physiological performance. This study, carried out over two consecutive wheat growth seasons (rabi seasons of 2012–13 and 2013–14), examined how environmental factor particular soilwhich was influenced by agricultural management, broughtto alter physiological contents in leaves of wheat during grain filling period. Proline accumulation decreased significantly from conventional agricultural practices (scenario 1) to conservation agricultural practices (scenario 3). There were significant differences occurred between all three scenarios of agricultural management. The lowest average proline concentration in two consecutive years was found 10.97 (μg proline/fresh weight) in flag leaves of wheat during grain filling stage grown under conservation agriculture practices, while highest concentration was found 33.97, grown in conventional agricultural practices. It was concluded that proline was reliable biomarker of the environmental stress imposed on crop, thus allowing us to establish stress onsets for quality yield of conservation agriculture grown wheat.