Floral initiation is required for sexual reproduction in angiosperm plants, and has a significant impact on crop yields. In cultivated strawberry, the molecular basis of floral initiation is poorly understood and most studies have focused on a single genotype grown under controlled conditions. To gain more insight into this process, we conducted a study under natural conditions in two countries using two seasonal flowering cultivars. We focused on the early steps of floral initiation by using samples spanning key developmental stages of the shoot apical meristem. The analysis of differential gene expression in leaf and terminal bud tissues revealed enrichment for genes involved in carbohydrate metabolism and phytohormone pathways in leaves. Other protein classes that were enriched during early floral initiation were associated with cytoskeleton organization, cell cycle regulation, and chromatin structure. We also identified genes associated with the photoperiodic pathway, well-characterized floral integrators such as TFL1 and SOC1, and several linked to phytohormone regulation, such as XTH23, PP2 and EIN3.