The hippocampus plays a critical role in pattern separation, a process that allows the discrimination of similar experiences, essential for episodic memory. This review summarizes evidence from electrophysiological recordings, lesion studies, immediate early gene imaging, transgenic mouse models, and human functional neuroimaging, showing that specific hippocampal subfields, particularly the dentate gyrus (DG) and CA3, are involved in this process. The DG is thought to perform pattern separation, while CA3 is involved in pattern completion. The DG granule cells are especially effective at pattern separation, and their activity is influenced by factors such as aging and adult neurogenesis. Neurogenesis in the DG may support pattern separation, although the exact mechanisms remain unclear. Lesion studies in rats show that DG is necessary for spatial pattern separation, and impairments in DG function lead to deficits in pattern separation. Similarly, CA3 is involved in pattern completion, as evidenced by studies showing that CA3 lesions impair recall of place-object associations. Aging is associated with impairments in pattern separation, particularly in the DG and CA3, which may contribute to episodic memory deficits in older adults. Neurogenesis in the DG may play a facilitative role in pattern separation, but the exact contributions of newborn versus mature granule cells are still under investigation. Human fMRI studies have shown that DG/CA3 activity is linked to pattern separation, with DG showing stronger pattern separation signals. However, the exact mechanisms of pattern separation and its relationship to other memory processes remain complex and require further study. The role of the hippocampus in pattern separation is also linked to other cognitive functions, such as visual perception, object recognition, and olfactory discrimination. The review highlights the importance of understanding the computational framework of pattern separation and its implications for memory function across species and age. Despite significant progress, many questions remain about the exact mechanisms and roles of pattern separation in the hippocampus and its relationship to other cognitive processes.The hippocampus plays a critical role in pattern separation, a process that allows the discrimination of similar experiences, essential for episodic memory. This review summarizes evidence from electrophysiological recordings, lesion studies, immediate early gene imaging, transgenic mouse models, and human functional neuroimaging, showing that specific hippocampal subfields, particularly the dentate gyrus (DG) and CA3, are involved in this process. The DG is thought to perform pattern separation, while CA3 is involved in pattern completion. The DG granule cells are especially effective at pattern separation, and their activity is influenced by factors such as aging and adult neurogenesis. Neurogenesis in the DG may support pattern separation, although the exact mechanisms remain unclear. Lesion studies in rats show that DG is necessary for spatial pattern separation, and impairments in DG function lead to deficits in pattern separation. Similarly, CA3 is involved in pattern completion, as evidenced by studies showing that CA3 lesions impair recall of place-object associations. Aging is associated with impairments in pattern separation, particularly in the DG and CA3, which may contribute to episodic memory deficits in older adults. Neurogenesis in the DG may play a facilitative role in pattern separation, but the exact contributions of newborn versus mature granule cells are still under investigation. Human fMRI studies have shown that DG/CA3 activity is linked to pattern separation, with DG showing stronger pattern separation signals. However, the exact mechanisms of pattern separation and its relationship to other memory processes remain complex and require further study. The role of the hippocampus in pattern separation is also linked to other cognitive functions, such as visual perception, object recognition, and olfactory discrimination. The review highlights the importance of understanding the computational framework of pattern separation and its implications for memory function across species and age. Despite significant progress, many questions remain about the exact mechanisms and roles of pattern separation in the hippocampus and its relationship to other cognitive processes.