To clarify the systematic relationships and evolutionary history of South American deer, we conducted a comprehensive phylogenetic analysis using representative species of all of the genera of Neotropical deer. Our results revealed high levels of molecular and cytogenetic divergence between groups of morphologically similar species of brockets (Mazama), and suggest a polyphyletic origin. At least eight ancestral forms of deer invaded South America during the late Pliocene (2.5-3 MYA), and members of the red brockets had an independent early explosive diversification soon after their ancestor arrived there, giving rise to a number of morphologically cryptic species.