Long-lived conifers are vulnerable to climate change because classical evolutionary processes are slow in developing adaptive responses. Therefore, the capacity of a genotype to adopt different phenotypes is important. Gene expression is the primary mechanism that converts genome-encoded information into phenotypes, and DNA methylation is employed in the epigenetic regulation of gene expression. We investigated variations in global DNA methylation and gene expression between three Scots pine (Pinus sylvestris L.) populations located in northern and southern Finland using mature seeds. Gene expression levels were studied in six DNA methyltransferase (DNMT) genes, which were characterized in this study, and in 19 circadian clock genes regulating adaptive traits. In embryos, expression diversity was found for three DNMT genes, which maintain DNA methylation. The expression of two DNMT genes was strongly correlated with climate variables, which suggests a role for DNA methylation in local adaptation. For adaptation-related genes, expression levels showed between-population variation in 11 genes in megagametophytes and in eight genes in embryos, and many of these genes were linked to climate factors. Altogether, our results suggest that differential DNA methylation and gene expression contribute to local adaptation in Scots pine populations and may enhance the fitness of trees under rapidly changing climatic conditions.