The present study investigated the dry matter yield (DMY) and arbuscular mycorrhizal (AM) colonization (internal hyphae, arbuscules, and vesicles) of two genotypes of zoysia grass (Zoysia japonica strain Aso and cv. Asagake) at three rates (10, 20 and 40 g N/m2/year) of application of digested slurry (DS) or mineral fertilizer. Soil chemical properties (available phosphorus concentration, pH and electrical conductivity (EC)) were also measured as factors associated with AM colonization. The fertilizer type (DS v. mineral fertilizer) had an effect on DMY depending on the plant genotypes but had little or no effect on AM colonization, and soil chemical properties, indicating that DS can be used as a substitute for mineral fertilizer; however, it is necessary to apply it taking into account the ratio of inorganic nitrogen (N). However, heavy DS application decreased colonization by internal hyphae. Furthermore, even at the lowest rate, the continuous application of DS decreased colonization by arbuscules and vesicles in both genotypes, which were associated with decreasing soil pH and increasing EC. The results suggest that zoysia grass should be fertilized with DS at a rate of <10 g N/m2/year in order to achieve a continuous association with AM fungi, although this range of N application is sub-optimal for zoysia grass production.