The evolution of signals and reproductive traits involved in the pre-mating recognition has been in focus of abundant research in several model species group such as bumblebees (genus Bombus). However, the most studied bumblebee reproductive trait, the male cephalic labial gland secretions (CLGS), remains unknown among bumblebee species from South America. In this study we investigated the CLGS of five South American bumblebees of the subgenera Thoracobombus (Bombus excellens, B. atratus) and Cullumanobombus (B. rubicundus, B. hortulanus and B. melaleucus) comparing their secretions to those of European closely related species. Our results show an obvious interspecific differentiation in all subgenera. The interspecific differentiation in Thoracobombus involves different compounds with a large abundance (main compounds) while all Cullumanobombus share the same main components. This suggests that in Cullumanobombus subgenus differentiation in minor components could lead to species discrimination.