AbstractThe phylogeny of the very diverse Neotropical fish family Characidae has been the subject of several recent contributions based on morphological characters, molecular data or both in combined analyses. In cases of conflict between these kinds of data, resolution by combined analyses most often tends to agree with the molecular evidence, given the disproportionate number of characters it contains in comparison with morphological datasets. This happens especially after the advent of massive DNA sequencing methods. In this contribution, we present the most comprehensive set of characters from sperm and spermiogenesis of the Characidae. Since these traits are not expected to be functionally correlated with the general morphology or molecular markers, we consider them a third source of data. We provide a phylogenetic analysis from a combined dataset of seven molecular markers (6444 characters), general morphology (520 characters) and reproductive features (94 characters) coded for 165 species of characiform fishes. Parsimony analyses were done under extended implied weighting under 30 different combinations of weighting schemes and strengths. Most parsimonious trees from two different weighting conditions were selected as representative samples of the obtained topologies, in order to evaluate the performance of the reproductive characters. One of these hypotheses is more conservative regarding the currently accepted phylogenies and the other is the most parsimonious tree that we found as the best correlated with the morphological data. Reproductive characters are shown to be more homoplastic than general morphology and DNA, but provided synapomorphies for 23–24 nodes that had no morphological synapomorphies, justifying their use in phylogenetic analyses. Also, in combination with data from general morphology and considering details of the phylogenetic analysis, they showed to have the potential to challenge well‐established hypotheses based on molecular data.