The evolutionary origin of extant species in the Neotropics, one of the most biodiverse regions of the world, has been widely debated. One hypothesis is that neotropical species emerged primarily during the Quaternary (the last approximately 2 million years), favoured by alternating glacial/interglacial climates. An opposite view proposes an older Tertiary origin linked primarily to palaeogeographical changes. Here, a thorough review of the available literature on DNA molecular dating shows that the Tertiary-Quaternary debate no longer makes sense. Indeed, the > 1400 neotropical species whose origin has been dated have appeared in a continual fashion since the late Eocene/early Oligocene (approximately 39 million years before present) to the Quaternary. Palaeogeographical mechanisms of speciation are relatively well accepted, but diversification processes linked to climate are still controversial. These results are important to unravel both the origin of present-day biodiversity patterns at both local and global scales and the genetic and environmental mechanisms involved, which are two crucial aspects for suitable biodiversity conservation strategies.