Unbiased stereological analysis of 5XFAD mice reveals significant neuron loss in cortical Layer 5 at 9 and 12 months of age. Brains from age-matched non-transgenic littermate (non-Tg) and 5XFAD female mice were sectioned at 30 μm and stained with cresyl violet and imaged by light microscopy. Large pyramidal neurons were then counted in Layer 5 cortex using an optical fractionator. (A) Large pyramidal neuron numbers in non-transgenic and 5XFAD Layer 5 cortex from mice aged 4–12 months. At 9 and 12 months there is a statistically significant decrease in the number of large pyramidal neurons in 5XFAD compared to non-transgenic mice (* p<0.05, ** p<0.01; n = 5 mice per group). 5XFAD mice exhibited a progressive decrease in Layer 5 large pyramidal neuron number, which at 12 months represented a 24.8% loss compared to non-transgenic mice. (B) 6 month 5XFAD mice are separable into two subgroups: those with normal neuron counts (6Mo 5XFAD high) and those with low neuron counts indicating neuron loss (6Mo 5XFAD low). The 5XFAD low count group showed a statistically significant decrease in neuron number compared to age matched non-transgenic and 5XFAD normal count groups (* p<0.05, ** p<0.01, n = 5 non-Tg, n = 10 net 6 month 5XFAD).