[Objective] Pooling of multiple samples is widely used in studying general patterns of microbial communities that are heterogeneously structured in space. Pooling strategies and the number of sequence reads generate biases in the description of diversity and community structure of root-associated fungi. Therefore, we developed a molecular toolbox for fast and accurate identification of the root-associated fungal community of Rododendron species. [Methods] Multiple root samples of R. lutescens and R. bureavii were collected for DNA extraction. Effects of two different pooling strategies, i.e. pooling samples prior to vs. post PCR, on fungal species composition were studied by comparing results within host species. [Results] Species richness and Shannon-Wiener index of fungal communities of clone library constructed by pooling samples after PCR were higher than that of pooling prior to PCR. High frequency fungal species were detected by both pooling strategies, whereas infrequent species detected by the two strategies differed. Notably, the prior to PCR pooling strategy effectively alleviated the unwanted amplification of host plant sequences when fungal specific primer ITS1f and ITS4 were used. Accumulation curves of fungal species suggested that sequencing at least 50 clones can fully reflect species composition of clone library of the two Rhododendron root-associated fungal community. [Conclusion] Clone library constructed by post PCR pooling of samples is better in providing accurate views of fungal diversity and community structure of Rhododendron species.