The mechanism of self-organization of quantum dots (QDs) during the growth of InGaAs/GaAs multilayers on GaAs (1 0 0) was investigated with cross-sectional transmission electron microscopy (XTEM), and double-crystal X-ray diffraction (DCXD). We found that the QDs spacing in the first layer can affect the vertical alignment of QDs. There seems to exist one critical lateral QD spacing, below which merging of QDs with different initial size is found to be the dominant mechanism leading to perfect vertical alignment. Once the critical value of QDs spacing is reached, the InGaAs QDs of the first layer are simply reproduced in the upper layers. The X-ray rocking curve clearly shows two sets of satellite peaks, which correspond to the QDs superlattice, and multi-quantum wells (QW) formed by the wetting layers around QDs.