Mutation and recombination drive the evolution of most pathogens by generating the genetic variants upon which selection operates. Those variants can, for example, confer resistance to host immune systems and drug therapies or lead to epidemic outbreaks. Given their importance, diverse evolutionary studies have investigated the abundance and consequences of mutation and recombination in pathogen populations. However, some controversies persist regarding the contribution of each evolutionary force to the development of particular phenotypic observations (e.g., drug resistance). In this study, we revise the importance of mutation and recombination in the evolution of pathogens at both intra-host and inter-host levels. We also describe state-of-the-art analytical methodologies to detect and quantify these two evolutionary forces, including biases that are often ignored in evolutionary studies. Finally, we present some of our former studies involving pathogenic taxa where mutation and recombination played crucial roles in the recovery of pathogenic fitness, the generation of interspecific genetic diversity, or the design of centralized vaccines. This review also illustrates several common controversies and pitfalls in the analysis and in the evaluation and interpretation of mutation and recombination outcomes. © 2017 Elsevier B.V.