The hippocampus is one of the earliest brain regions affected in Alzheimer's disease (AD) and tests of hippocampal function have the potential to detect AD in its earliest stages. Given that the hippocampus is critically involved in allocentric spatial memory, this study applied a short test of spatial memory, the 4 Mountains Test (4MT), to determine whether test performance can differentiate mild cognitive impairment (MCI) patients with and without CSF biomarker evidence of underlying AD and whether the test can distinguish patients with MCI and mild AD dementia when applied in different cultural settings. Healthy controls (HC), patients with MCI, and mild AD dementia were recruited from study sites in UK and Italy. Study numbers were: HC (UK 20, Italy 10), MCI (UK 21, Italy 14), and AD (UK 11, Italy 9). Nineteen UK MCI patients were grouped into CSF biomarker-positive (MCI+, n = 10) and biomarker-negative (MCI-, n = 9) subgroups. Behavioral data were correlated with hippocampal volume and cortical thickness of the precuneus and posterior cingulate gyrus. Spatial memory was impaired in both UK and Italy MCI and AD patients. Test performance additionally differentiated between MCI+ and MCI- subgroups (P = 0.001). A 4MT score of ≤8/15 was associated with 100% sensitivity and 90% specificity for detection of early AD (MCI+ and mild AD dementia) in the UK population, and with 100% sensitivity and 50% specificity for detection of MCI and AD in the Italy sample. 4MT performance correlated with hippocampal volume in the UK population and cortical thickness of the precuneus in both study populations. In conclusion, performance on a hippocampus-sensitive test of spatial memory differentiates MCI due to AD with high diagnostic sensitivity and specificity. The observation that similar diagnostic sensitivity was obtained in two separate study populations, allied to the scalability and usability of the test in community memory clinics, supports future application of the 4MT in the diagnosis of pre-dementia due to AD.