A central problem in evolutionary biology is identifying factors that promote the evolution of reproductive isolation. Among mosses, biogeographic evidence indicates that the potential for migration is great, suggesting that biological factors other than geographic isolation may be critical for speciation in this group. The moss Physcomitrella patens (Funariaceae) has long been used as a model for interspecies hybridization and has recently emerged as an important model system for comparative genomics. We report genealogical analyses of six loci from several populations of P. patens and related species in the genus Physcomitrium. These results unambiguously indicate that the so-called genus Physcomitrella arose at least three times from distinct ancestors within the genus Physcomitrium. In spite of the evidence for natural hybridization in the Physcomitrella–Physcomitrium complex, genealogical and experimental hybridization data indicate that the taxonomically defined species are reproductively isolated. However, these analyses suggest that Physcomitrium eurystomum was formed from a hybridization event between two early diverging lineages in the complex, and that the ancestral population size of these lineages was much smaller than the current population sizes. We discuss these findings in the context of the inferred mating system in the Physcomitrella–Physcomitrium complex and patterns of speciation and diversification.