Microglia have been identified as key players in Alzheimer's disease pathogenesis, and other neurodegenerative diseases. Iba1, and more specifically TMEM119 and P2RY12 are gaining ground as presumedly more specific microglia markers, but comprehensive characterization of the expression of these three markers individually as well as combined is currently missing. Here we used a multispectral immunofluorescence dataset, in which over seventy thousand microglia from both aged controls and Alzheimer patients have been analysed for expression of Iba1, TMEM119 and P2RY12 on a single-cell level. For all markers, we studied the overlap and differences in expression patterns and the effect of proximity to β-amyloid plaques. We found no difference in absolute microglia numbers between control and Alzheimer subjects, but the prevalence of specific combinations of markers (phenotypes) differed greatly. In controls, the majority of microglia expressed all three markers. In Alzheimer patients, a significant loss of TMEM119+-phenotypes was observed, independent of the presence of β-amyloid plaques in its proximity. Contrary, phenotypes showing loss of P2RY12, but consistent Iba1 expression were increasingly prevalent around β-amyloid plaques. No morphological features were conclusively associated with loss or gain of any of the markers or any of the identified phenotypes. All in all, none of the three markers were expressed by all microglia, nor can be wholly regarded as a pan- or homeostatic marker, and preferential phenotypes were observed depending on the surrounding pathological or homeostatic environment. This work could help select and interpret microglia markers in previous and future studies.@en