The commercial strawberry (Fragaria x ananassa) resistance gene Rpfl conferring resistance to various isolates of Phytophthora fragariae, was mapped using 7 RAPD markers. A DNA fragment representing a RAPD marker linked to susceptibility was cloned, sequenced and converted into a sequence characterized amplified region (SCAR) marker. Next, SCAR primers to the resistant allele (SCAR-R) were developed based upon a deletion region between susceptible and resistant plants. The SCAR-R primers were tested on a F1 progeny of 60 plants in which Rpf1 segregated in a 1:1 ratio. SCAR-R primers amplified DNA from resistant plants only. The SCAR-R primers and three RAPD primers linked to and surrounding the Rpfl gene were used to screen numerous European and North American strawberry genotypes. The molecular data supported the currently known plant resistance testing and indicates that the resistance gene region has been conserved in most crosses. The linkage to Rpfl with these markers was lost in some genotypes due to crossing-over, for example the subsequent generations of ‘Stelemaster’. The accuracy of these molecular markers for the detection of the gene demonstrates that they can be used in a marker-assisted strawberry breeding programme.