Infections frequently contain multiple strains (genotypes) of the same pathogen, yet they are still usually treated as uniform entities. In this Review, we discuss problems with inconsistent definition of the term “strain” and review the prevalence and implications of multiple-strain infections. Up to now, multiple-strain infections have been shown unambiguously in 51 human pathogens (and 21 non-human ones) and are likely to arise in most pathogen species. In human pathogens, multiple-strain infections usually reach considerable frequencies (median 11·3%, mean 21·7% of infections), which are certainly underestimated in many cases because of technical limitations of detection. For many diseases, the importance of multiple-strain infections is still unclear, but theoretical work and experimental results from animal models suggest a broad range of clinically relevant effects. Multiple-strain infections can affect host immune responses and our ability to prevent and treat infection efficiently. Competition and mutualism between strains change pathogen and disease dynamics and promote pathogen evolution. Co-infection enables gene transfer among strains. Taking multiple-strain infections into account will improve our understanding of host-pathogen interactions and disease dynamics, and will provide a basis for novel control approaches.