Abstract: 【Objective】Soil eukaryotes are key indicator organisms for soil health in ecosystems, and changes in their diversity and community structure can effectively reflect the evolution of soil quality. In high-throughput sequencing-based studies on eukaryotic diversity, the selection of amplification primers directly affects the number of sequences of detected taxa, thereby determining the accuracy of biodiversity assessment. However, the impact of primer selection on the assessment of soil eukaryotic diversity remains timidly explored.【Method】 This study focused on soils from seven typical urban land use types in Ningbo City. Amplification was only conducted for two pairs of widely used 18S-rRNA gene V4 region primers (NF1F_18Sr2bR, TAReuk454FWD1F_TAReukREV3R). The study systematically compared the effects of different primers on the assessment of soil eukaryotic community composition and diversity, and analyzed the differences between two bioinformatic methods: Amplicon sequence variants (ASVs) and operational taxonomic units (OTUs). 【Result】That the proportion of amplification in eukaryotes for the NF1F_18Sr2bR primer was significantly higher than that of TAReuk454FWD1F_TAReukREV3R. Different primers exhibited a preference for specific soil eukaryotic taxonomic groups. Specifically, at the ASVs level, the NF1F_18Sr2bR primer preferred fungi, protozoa, nematoda, arthropoda, and annelida; at the OTUs level, the TAReuk454FWD1F_TAReukREV3R primer preferred protozoa and arthropoda, while the NF1F_18Sr2bR primer preferred fungi, nematoda, and annelida. Among the primers, NF1F_18Sr2bR was more appropriate for detecting rare species. The rare species of fungi, nematoda, and annelida amplified by this primer accounted for 12.09%, 38.31%, and 58.33% of their total sequences, respectively. In contrast, TAReuk454FWD1F_TAReukREV3R was more suitable for detecting shared species, as it detected 804 shared species OTUs across different land use types, which was higher than that detected by the other primer. Both primer selection and analytical methods collectively determine the differences in α-diversity assessment, but they do not determine the variations in β-diversity or the effects of environmental factors on community structure. In terms of α-diversity, for both primer pairs, the differences in α-diversity among different land uses were greater at the ASV level than at the OTU level. With respect to β-diversity, the explanatory power of the OTUs level for community diversity was higher than that of the ASVs level. 【Conclusion】This study revealed the critical impact of primer selection on the assessment of soil eukaryotic diversity. In future studies, primers and analytical methods should be selected appropriately based on target taxa and research objectives to ensure the accuracy of community structure and diversity assessment.