We have identified a reliable set of multiplexed microsatellite (SSR) markers for the genotyping of strawberry cultivars and their octoploid progenitors. Over 100 SSRs were screened in two F. × ananassa genotypes and from these, 32 that showed promise for genotyping were selected for further analysis. These SSRs were used to screen a set of 16 strawberry cultivars and a set of fingerprints were produced. Those SSRs that produced reliable, reproducible and easy to interpret fingerprints, that could also distinguish readily between the 16 strawberry cultivars screened, and which could be conveniently included in three multiplex reactions, were selected to form the genotyping set. The genotyping set, consisting of 10 previously-reported SSRs was used to fingerprint a total of 56 cultivated strawberry, and four octoploid Fragaria species accessions. The SSRs used could reliably distinguish between all 60 genotypes surveyed, including sibling cultivars derived from the same parental lines. The primers could be combined for multiplex PCR and represent a useful and convenient genotyping set for Fragaria that will permit fingerprinting data to be shared between laboratories.