Structural hippocampal abnormalities are common in many neurological and psychiatric disorders, and even variation within the normal range is related to cognitive performance and other complex phenotypes such as stress sensitivity. Hippocampal subregions are increasingly studied, as automated algorithms have become available for mapping and volume quantification. In the context of the ENIGMA Consortium, several Disease Working Groups are using the FreeSurfer software to analyze hippocampal subregion (subfield) volumes in patients with major depressive disorder, bipolar disorders, posttraumatic stress disorder, and other conditions, along with data from matched controls. In this overview, we explain the algorithm’s principle, summarize measurement reliability studies, and demonstrate two additional aspects (subfield autocorrelation and volume/reliability correlation) with illustrative data. We then explain the rationale for a standardized hippocampal subfield segmentation quality control (QC) procedure for improved pipeline harmonization. To guide researchers to make optimal use of the algorithm, we discuss how global size and age effects can be modelled, how QC steps can be incorporated and how subfields may be aggregated into composite volumes. This discussion is based on a synopsis of 162 published neuroimaging studies (01/2013–12/2019) that applied the FreeSurfer hippocampal subfield segmentation in a broad range of domains including cognition and healthy aging, brain development and neurodegeneration, affective disorders, schizophrenia, childhood trauma and posttraumatic stress disorder, inflammatory and systemic disease, alcoholism, stress regulation, neurotoxicity, as well as assessments of heritability, effects of candidate genes, genome-wide association studies and epigenetic effects. Lastly, we highlight points where FreeSurfer-based hippocampal subfield studies may be optimized.