Cell adhesion plays an essential role in development and homeostasis, but is also a key regulator of many diseases such as cancer and immune dysfunction. Numerous studies over the past three decades have revealed a wealth of information detailing signalling molecules required for cell adhesion to two-dimensional surfaces. However, in vivo many cells are completely surrounded by matrix and this will very likely influence the size, composition and dynamics of adhesive structures. The study of adhesion in cells within three-dimensional environments is still in its infancy, thus the role and regulation of adhesions in these complex environments remains unclear. The recent development of new experimental models coupled with significant advances in cell imaging approaches have provided platforms for researchers to begin to dissect adhesion signalling in cells in 3D matrices. Here we summarise the recent insights in cell adhesion formation and regulation in 3D model systems and the imaging approaches used to analyse these events.