The bed nucleus of the stria terminalis (BNST)—a small gray matter region located in the basal forebrain—has been implicated in both anxiety and addiction based on compelling evidence from rodent and non-human primate studies. However, the BNST's small size and proximity to other gray matter regions has hindered non-invasive study in human subjects using standard neuroimaging methods. While initial studies have benefitted from a BNST mask created from a single human subject using a 7 T scanner, individual variability is likely—especially in patient populations—thus a manual segmentation protocol is needed. Here we report on the development of a reliable manual segmentation protocol performed on 3 T MRI images using a scanning sequence that provides high gray matter/white matter/cerebrospinal fluid contrast. Inter- and intra-rater reliabilities, measured in 10 healthy individuals, demonstrate that the protocol can be reliably implemented (intra-rater Dice similarity coefficient≥0.85, inter-rater≥0.77). This BNST tracing protocol provides the necessary foundation for future 3 T MRI studies of the BNST in healthy controls and patient populations.