The winter hardiness of strawberry varieties used in perennial production systems varies greatly. Still, little information is available on how plant metabolism adapts to cold and freezing temperatures under natural temperature and light conditions. In order to examine the hardening process of overwintering meristematic tissue in Fragaria ananassa, crown samples of field-grown var. ‘Polka’ and ‘Honeoye’ were consecutively collected over a period of 15 weeks, i.e. from the end of the season (week 35/ end August) until midwinter (week 50/ December). Samples were subjected to qGC MS metabolite profiling to assess the reconfiguration of central metabolism, and characterize the regulation of selected compatible solutes. Besides changes in amino acid patterns (glutamic acid, aspartic acid, and asparagine), monosaccharide levels (fructose) increased strongly in ‘Honeoye’ (180 fold compared to start control) towards the end of the acclimation period. In contrast, ‘Polka’ showed a concentration peak (36-fold) in week 47 and a decline towards week 50. Also sucrose levels were steadily increased throughout the cold hardening period with averagely 6-fold higher levels in ‘Honeoye’ compared to ‘Polka’, thus underscoring cultivar-dependent differences. However, both varieties showed a decline in sucrose levels after week 47. Particularly, the raffinose pathway was affected leading to strongly and transiently increased levels of the precursor galactinol (week 42/ mid-October) and the trisaccharide raffinose (weeks 43 to 47/ end October to mid-November). While galactinol biosynthesis was earlier induced in ‘Polka’ (week 38) compared to ‘Honeoye’ (week 39), subsequent raffinose production was delayed in ‘Polka’ (week 47) compared to ‘Honeoye’ (week 45). Major metabolic changes in both varieties coincided with a decrease in day length below 14 h in mid-September, and a consistent drop below 10°C average day temperature by the end of September