This study aimed to analyze spatial patterns of soil ectomycorrhizal fungal (EMF) communities at the local scale in a subtropical pine-oak forest located in the Nearctic-Neotropical transition in central Mexico, to underpin biodiversity conservation strategies in forest fragments of this region. We used a spatially-explicit nested square sampling design with the same sampling representativeness at all scales and replicated three times. We detected 674 EMF OTUs within 19,200 m² and 65 OTUs on average per sample. Seventy percent of OTUs were detected in only 1–4 samples. Average community similarity was below 5%, showed minor change within 14 and 339 m distance and increased with the spatial grain used to compare the data. We found a high species-area relationship and beta diversity coefficients for soil fungi indicating that, at the local scale, increasing area by a constant factor of four represented an increase in OTU richness by a factor of two.