Off-season strawberry production may diversify the yield, thereby increasing costs, but the environmental conditions are a limiting factor. This experiment aimed to study the effects of vernalization on off-season flowering and gene expression in sub-tropical strawberry cv. Pharachatan 80. The factorial (2 × 2) + 1 in a completely randomized design was used in this study. Factor A was the vernalization temperatures: 2 °C and 4 °C. Factor B was the vernalization periods: 1 week and 2 weeks, compared with non-vernalization (control). The expression profile of genes was determined after vernalization treatments. The results revealed an interaction between the two factors on the number of days it took the plants to bloom, the percentage of flowering, the number of inflorescences, the number of flowers per inflorescence and the number of flowers per plant, whereas the number of first flower bloom days, inflorescence length and flower size were not affected by the interaction between the two factors. Strawberry plants vernalized for 1 and 2 weeks at 2 °C showed earlier flowering (21.4 and 23.1 days, respectively) than did those vernalized at 4 °C (24.9 and 25.7 days, respectively). On the other hand, non-vernalized strawberry plants took longer to bloom, at 62.2 days. Strawberry plants vernalized at 2 °C for 2 weeks had the highest percentage of flowering, number of inflorescences, number of flowers per inflorescence and number of flowers per plant. The analysis on gene expression showed that VRN5, SOC1 and FT genes were upregulated after vernalization at 2 °C for 2 weeks, whereas gene expression of the control treatment was not detected. This study demonstrates that vernalization treatment could induce off-season flowering in sub-tropical strawberry cv. Pharachatan 80 by activating flowering genes.