This study investigates the vertical inter-QDs spacing (VIDS) in a coupled bilayer quantum dots (CBQD) heterostructure using cross-sectional High Resolution Transmission Electron Microscopy (HRTEM). Simultaneously, we also examined the interrelationship of VIDS with the strain energy inside the CBQD stack. As a continuation, the CBQD heterostructure were further explored from different aspects of growth parameters, such as the deposition of ultrathin GaAs spacer layer of 4.0–4.5 nm between the seed and active layer of QDs, the effect of larger monolayer coverage (3.2 ML) of seed layer QDs, and the optimization of growth rates for the CBQDs. Moreover, we present a model technique to characterize the in-plane 2θχ/Φ of InAs QDs and GaAs at (002) and (004) planes in order to analyze the strain. This study was the first of its kind to look at the formation of self-assembled In(x)Ga(1-x)As layer at the interface across the ultrathin GaAs spacer and InAs QDs, verified with HRTEM images. Smaller size of QDs formed in the seed layer led to the formation of a non-uniform self-assembled In(x)Ga(1-x)As layer. The problem of non-uniformity of In(x)Ga(1-x)As layer was resolved by increasing the seed layer monolayer coverage from 2.5 to 3.2 ML. The percentage of gallium adatoms inter-diffused into the outer surface of InAs QDs to form the self-assembled In(x)Ga(1-x)As layer was ∼31% for VIDS ∼1.4 ± 2 nm.