Extending the period of fruit production is a way to substantially increase crop yield in many fruit or ornamental species. In the cultivated octoploid strawberry (Fragaria x ananassa), the most consumed small fruit worldwide, fruit production season can be extended by selecting perpetual flowering (PF) cultivars. This trait is of considerable interest to growers and to the food industry. Four homoeologous loci controlling a single trait can be expected in such a complex octoploid species. However, we recently showed that the PF trait is under the control of the single dominant FaPFRU locus (Gaston et al., 2013), making it potentially amenable to marker assisted selection (MAS). Here, we report the successful use of a strategy, based on selective mapping using a reduced sample of individuals, to identify nine markers in close linkage to the FaPFRU allelic variant. Thus, this strategy can be used to fine map target homoeologous loci in other complex polyploid crop species. Recombinant analysis further enabled us to reduce the locus to a region flanked by two markers, Bx083_206 and Bx215_131, corresponding to a 1.1 Mb region in the diploid F. vesca reference genome. This region comprised 234 genes, including 15 flowering-associated genes. Among these, the FLOWERING LOCUS T (FT) is known to be a key activator of flowering. The close association between the PF trait and the FaPFRU flanking markers was validated using an additional segregating population and genetic resources. This study lays the foundation for effective and rapid breeding of PF strawberry cultivars by MAS. This article is protected by copyright. All rights reserved.