Mild Cognitive Impairment (MCI) is a transitional state between normal aging and dementia. Of the cognitive abilities that may decline in MCI (e.g., memory, language, executive functioning, visuospatial processing), memory is of particular interest for identifying individuals who may progress and develop Alzheimer's disease (AD). A particular mnemonic mechanism that declines in older adults is spatial pattern separation. Spatial pattern separation reduces the interference among spatially similar memory representations and increases the probability of accurate encoding and retrieval. Examining spatial pattern separation in MCI may provide insight into a specific memory mechanism that is adversely affected by MCI associated pathology. Pattern separation is facilitated by the dentate gyrus (DG) and the CA3 subregions of the hippocampus. Individuals with the clinical subtype amnestic MCI (aMCI) evidence hyperactivity in the DG and CA3 subregions as well as smaller DG and CA3 subregion volumes compared to individuals without MCI. Previous studies in humans have suggested that individuals with MCI are impaired when performing a non-spatial pattern separation task compared to controls; however, no study to date has examined whether individuals with MCI evidence impaired performance on a spatial pattern separation task. The current study used a spatial pattern separation task developed in our laboratory to examine whether individuals with MCI evidence spatial pattern separation deficits compared to age-matched controls. The task involved the presentation of a single circle on a computer screen and then after a delay, the presentation of two circles—one target circle in the same location as the original circle and one foil circle in a different location. Participants were asked to indicate which of the two circles was in the same location as the original circle. Four possible distances separated the edges of the target and foil circle: 0.0 cm, 0.5 cm, 1.0 cm, or 1.5 cm. Smaller distances between the target and foil circle were hypothesized to result in increased interference and heightened demand for spatial pattern separation. Data were analyzed using a 2 x 4 repeated measures ANOVA with group (MCI, control) as the between subjects variable and separation distance (0 cm, 0.5 cm, 1.0 cm, and 1.5 cm) as the within subject variable. Healthy older adults were predicted to perform significantly better on the spatial pattern separation task compared to individuals with an MCI diagnosis. However, the present analyses revealed no significant differences on the spatial pattern separation task between individuals diagnosed with MCI and healthy older adults. Although the present findings suggest that an MCI diagnosis may not adversely affect spatial pattern separation, future studies examining particular subtypes of MCI (e.g., aMCI) or MCI groups diagnosed using more conservative criteria are needed to further examine the effect of MCI on spatial pattern separation.