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Arbuscular mycorrhizal fungi (AMF) are important for plant nutrient and water acquisition. Much is known about how nutrient addition and environment affect AMF, but little is known about nutrient by environment interactions. We measured AMF colonization with nutrient additions and along an environmental gradient to assess these interactions. We measured AMF colonization in roots of little bluestem (Schizachyrium scoparium (Michx) Nash) with nutrient addition and across an environmental gradient. We assessed how AMF colonization changed across different fertilization treatments, and used ridge regression to determine nutrient, environment, and nutrient by environment interaction variables that predicted AMF colonization. The addition of nitrogen decreased AMF colonization, while mean annual temperature (MAT) and soil pH both positively predicted the percentage of AMF colonization in Schizachyrium scoparium. Additionally, we found an interaction term between MAT and phosphorus treatments that significantly affected percent AMF colonization. Our results show the importance of understanding environmental conditions on AMF as well as nutrient by environment interactions when assessing how AMF respond to nutrient addition. Here we present a full-factorial nutrient addition study along an environmental gradient to assess how AMF root colonization is influenced by abiotic factors in addition to nutrients.