Flow instabilities in liquid films impact coatings, lubrications, and microfluidics. The flow instability conditions are known for thick films where variations in viscosity, surface tension, mass density as well as fluid inertial and viscoelastic effects can destabilize the flow. In molecularly thin films, however, because of the intimate coupling of the flow properties to surface confined molecular conformation and dynamics, there may exist additional mechanisms that destabilize the flow. In this respect, a particularly interesting system is that of compressible monolayers wherein molecules change their conformation in response to variations of the film pressure. In such systems, the molecular conformation is coupled to the film thickness, hence the flow velocity, and the friction coefficient between the spreading monolayer and substrate. Here we report a new type of flow instability triggered by conformational transitions of brushlike macromolecules as they spread on a solid substrate. The conformational transition segregates the flowing monolayer into two conformationally different phases and simultaneously leads to an abrupt decrease in the monolayer thickness of the down-flow phase. By tracing the movement of individual molecules, we are able to follow the evolution of the instability pattern on the molecular level and to understand the underlying physical mechanism. This finding presents the first example of the Saffman-Taylor–type instability in compressible monolayers where the flow velocity is controlled by molecular conformation. The proposed mechanism is general enough to be observed in other monolayer systems wherein pressure-induced conformational transitions could occur.