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Arbuscular mycorrhizal fungi (AMF) spore density and root colonization are considered sensitive to host species and abiotic factors such as climate and soil. However, there is a knowledge gap about how fragmented native forest remnants might contribute to AMF conservation, what is the AMF spore density and root colonization, and to what extent climate change, particularly warming, might impact AMF. The aim of the study was to quantify the AMF spore density and root colonization along altitudinal gradients in three agro-ecological zones of nine church forests in northern Ethiopia. Data were collected from 45 plots. All the surveyed church forest species were colonized by AMF. However, we found a significant (p < 0.05) decrease in root colonization and AMF abundance in forests at high elevation. The topsoil had significantly (p < 0.05) higher root colonization and AMF abundance than subsurface soil. We found strong negative correlations between altitude and both spore density and root colonization and soil fertility. While we cannot separate whether spore density was temperature or soil limited, we can demonstrate the importance of conserving certain tree species, particularly Ficus species, which harbor high spore densities, in both lowland and midland church forests. In the highland, no Ficus species were found. However, Hagenia abyssinica, another Rosales, had the highest spore density in the highland ecoregion.