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N6-methyladenine (6mA) DNA modification has been detected in several eukaryotic organisms, where it plays important roles in gene regulation and epigenetic memory maintenance. However, the genome-wide distribution patterns and potential functions of 6mA DNA modification in woodland strawberry (Fragaria vesca) remain largely unknown. Here, we examined the 6mA landscape in the F. vesca genome by adopting single-molecule real-time sequencing technology and found that 6mA modification sites were broadly distributed across the woodland strawberry genome. The pattern of 6mA distribution in the long non-coding RNA was significantly different from that in protein-coding genes. The 6mA modification influenced the gene transcription and was positively associated with gene expression, which was validated by computational and experimental analyses. Our study provides new insights into the DNA methylation in F. vesca.