Understanding genetic structure and diversity information is critical for genetic association studies. In the octoploid cultivated strawberry (Fragaria×ananassa), genetic analyses were focussed on diversity, whereas genetic structure has been poorly explored. This study investigated the genetic structure in a genetic resources collection representing a wide range of the octoploid strawberry cultivars released mainly by North America and western and southern Europe, at different breeding periods and with various pedigrees. The relationship between varieties was examined using 23 microsatellite (simple sequence repeat, SSR) markers. Eight SSR markers were diploid, useful for cultivar discrimination with polymorphic information content (PIC) values between 0.29 and 0.74. Bayesian analyses of genetic structure identified four subpopulations. Three of them, American and modern northern European cultivars (AMNECs), American and modern southern European cultivars (AMSECs) and old European cultivars (OECs), reflected the European breeding history of the cultivated octoploid strawberry. The fourth subpopulation, ‘Intermediate’ group cultivars (IGCs), comprised various origins including OECs that were introgressed with wild species such as Fragaria chiloensis or Fragaria moschata. The OEC group gathered cultivars dating before 1960s, forming the most homogenous and stable subpopulation. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on modified Nei and Li distance confirmed the separation of the AMSEC, AMNEC and OEC groups. In addition, significant differences were observed among the four subpopulations (AMNEC, AMSEC, OEC, IGC), with high variability within groups and between AMSEC and IGC. Our work underlined that the structure within the studied collection was mainly explained by the pedigree and the year of release than the geographical origin of cultivars. In addition, the important loss of diversity observed in the modern European cultivars and a trend towards using mainly American cultivars for breeding programmes led to the progressive abandonment of old European germplasm, which was revealed as a relative distinct and rich group. This European material should be protected and maintained, because it represents a potential source of original traits for broadening the genetic base of cultivated strawberry. In addition, diploid markers we identified can be used without ambiguity in phylogenetic and diversity studies, because they are genome-specific. This study is the first step for further association studies in strawberry.