Facial expression is a common mode of visual communication in mammals but especially so in primates. Rhesus macaques (Macaca mulatta) have a well-documented facial expression repertoire that is controlled by the facial/mimetic musculature as in all mammals. However, little is known about the musculature itself and how it compares with those of other primates. Here we present a detailed description of the facial musculature in rhesus macaques in behavioral, evolutionary and comparative contexts. Formalin-fixed faces from six adult male specimens were dissected using a novel technique. The morphology, attachments, three-dimensional relationships and variability of muscles were noted and compared with chimpanzees (Pan troglodytes) and with humans. The results showed that there was a greater number of facial muscles in rhesus macaques than previously described (24 muscles), including variably present (and previously unmentioned) zygomaticus minor, levator labii superioris alaeque nasi, depressor septi, anterior auricularis, inferior auricularis and depressor supercilii muscles. The facial muscles of the rhesus macaque were very similar to those in chimpanzees and humans but M. mulatta did not possess a risorius muscle. These results support previous studies that describe a highly graded and intricate facial expression repertoire in rhesus macaques. Furthermore, these results indicate that phylogenetic position is not the primary factor governing the structure of primate facial musculature and that other factors such as social behavior are probably more important. The results from the present study may provide valuable input to both biomedical studies that use rhesus macaques as a model for human disease and disorder that includes assessment of facial movement and studies into the evolution of primate societies and communication.