The emergence of resistance in combination therapy can be facilitated by treatment regimes where only one drug is active at certain periods of time, resulting in temporal effective monotherapy (e.g. structured treatment interruptions). This occurs because resistance mutations can arise in a stepwise instead of a concurrent manner when only one drug reaches an effective concentration. We propose that effective monotherapy can also occur at a spatial level because of discordant penetration of drugs into compartments of the body. Here, we present a mathematical model for the evolution of drug resistance in combination therapy when there is differential drug penetration. Our model shows that the presence of body compartments where only one drug in a combination regime penetrates to a therapeutic concentration strongly speeds up the evolution of multi-drug resistance. Resistance mutations are acquired in a stepwise manner through colonization of body compartments where the pathogen can survive without being resistant to all the drugs in the regime. These results offer a possible explanation for the evolution of single-drug resistance in the course of combination therapy. We compare our model results to clinical trials for antiretrovirals and antibiotics to identify cases where stepwise acquisition of mutations can be facilitated by differential drug penetration. Overall, our results suggest that the use of drugs with similar penetration profiles in combination therapy may prevent the evolution of multi-drug resistance.