BackgroundThe red brocket deer, Mazama americana, has at least six distinct karyotypes in different regions of South America that suggest the existence of various species that are today all referred to as M. americana. From an evolutionary perspective, the red brockets are a relatively recent clade that has gone through intense diversification. This study sought to prove the existence of post-zygotic reproductive isolation in deer offspring between distinct chromosome lineages. To achieve this, inter-cytotype and intra-cytotype crosses were performed, which resulted in both F1 hybrid (n = 5) and pure offspring (n = 3) in captivity.ResultsF1 females were analyzed in terms of their karyotypes, ovarian histology, estrous cycles and in vitro embryo production. Pure females presented parameters that were similar to those previously reported for M. Americana; however, the parameters for hybrid females were different. Two hybrids were determined to be sterile, while the remaining hybrids presented characteristics of subfertility.ConclusionsThe results support the existence of well-established reproductive isolation among the most distant karyotype lineages and elucidates the need to define all karyotype variants and their geographical ranges in order to define the number of species of red brocket.