Abstract: DNA methylation, as an epigenetic modification, plays a crucial role in the growth, development, and stress responses of plants. Whole genome bisulfite sequencing（WGBS） is considered as the "gold standard" for DNA methylation studies and is widely applied in the functional genomics research of both animals and plants. Although several analysis tools have been developed for WGBS, there has yet to be a comprehensive assessment of their analysis efficiency in plant data. This study conducted a thorough performance evaluation of six commonly used alignment methods（BSMAP, Bismark-bwt2-e2e, Bismark-his2, Abismal, BSSeeker2-bwt2-e2e, and BSSeeker2-bwt2-local） within four DNA methylation analysis tools. The evaluation encompassed aspects such as runtime efficiency, memory resource utilization, alignment quality, and identification of methylation sites by analyzing DNA methylation data from three major crops: Arabidopsis thaliana, Oryza sativa and Glycine max. The results indicate that although BSMAP requires larger memory requirements, it exhibits higher efficiency in terms of running speed, particularly when dealing with large-scale genomic data. Furthermore, BSMAP demonstrates high levels of alignment quality and methylated site identification, ensuring the accuracy and reliability of the results. The study emphasizes the need for researchers to make informed decisions when selecting alignment tools based on practical computational resources, research requirements, and the trade-off between running speed and memory consumption. This research provides valuable analytical guidance for scientists engaged in DNA methylation studies in the field of plants, contributing to improve research efficiency and result reliability. It holds significant scientific importance for a deeper analysis of DNA methylation in plant biology.