Abstract: Due to global warming, late spring coldness is a major agrometeorological disaster frequently occurring in wheat production. The wheat yield can be reduced by 30%–50% in severe cases. Rhizosphere microorganisms endow wheat with stress resistance, and variations in their community structures and ecological functions reflect the adaptability of wheat to environmental changes. Studying the impact of late spring coldness on the structure and diversity of fungal communities in the rhizosphere soil of wheat is important for understanding the disaster mechanisms of wheat in response to late spring coldness. This study used wheat cultivars of ‘Xinmai 26’ (XM, sensitive to late spring coldness) and ‘Yannong 19’ (YN, resistant to late spring coldness) as experimental subjects. A pot experiment was conducted to artificially simulate late spring coldness during the anther differentiation period (a sensitive stage) of young ear differentiation of wheat. With 10 ℃ as the control treatment (CK), two low-temperature stress treatments were set at 2 ℃ (T1) and −2 ℃ (T2). The rhizosphere soil was collected at the wheat flowering stage, and changes in the fungal community structure and diversity in the rhizosphere soil of different wheat cultivars were determined by the Illumina high-throughput sequencing platform. Additionally, the aboveground and belowground biomass of the wheat plants were measured, and the root-to-shoot ratio was calculated. Compared with CK, the aboveground and belowground dry matter weight of the two wheat cultivars at the flowering stage under late spring coldness decreased by 1.65%−12.22% and 15.05%−35.49%, respectively, and the root-to-shoot ratio increased by 15.79%−36.08%, which had a greater effect on XM than YN. Late spring coldness significantly increased the relative abundance of Ascomycota but significantly decreased the relative abundance of Mortierellales, Mortierellaceae and Mortierella, with a greater decrease in XM as the degree of stress increased. For XM, in T2 treatment, the relative abundance of Mortierella decreased by 54.70% compared with that of CK. The relative abundance of Agaricomycete fungi was significantly higher in YN than in XM under late spring coldness. The diversity analysis showed that late spring coldness significantly affected the diversity and richness of fungal communities in the wheat rhizosphere soil. The FUNGuild function prediction showed that the proportions of different ecological functional groups were affected by late spring coldness. The relative abundance of Endophytes was the highest, which decreased with the increase in late spring coldness. The wood saprotroph and plant pathogen were more abundant in XM_T2. In conclusion, late spring coldness decreased the aboveground and belowground biomass accumulation of wheat plants at the flowering stage, causing imbalanced growth and development of wheat roots and canopies, which was not conducive to yield. Late spring coldness significantly changed the community structure and diversity of wheat rhizosphere fungi; the degree of influence increased with an increase in the degree of stress, and the influence on XM was greater than that on YN. This study provides new insights into the disaster mechanism of late spring coldness in wheat from the perspective of rhizosphere microorganisms and provides theoretical support for exploring the response of the “root-soil-microbial” interaction system to late spring coldness from the perspective of the root-soil environment.