The improvement of strawberry (Fragaria × ananassa Duch.) quality such as flavour, day-neutrality, disease resistance, fruit firmness and colour through Australian Strawberry Breeding Program are the main concerns of local strawberry industries in recent years. This study aims to develop molecular markers closely linked to genes controlling flavour and day-neutrality traits by using a Subtracted Diversity Array for the initial marker discovery. In the present study, phenotypic characterization of two commercial strawberry cultivars (‘Albion’ and ‘Juliette’), and three promising breeding lines with intense flavour (07-102-41, 07-095-35 and 04-069-91) were evaluated using solid phase microextraction (SPME) coupled with two-dimensional gas chromatography (GC × GC) and time-of-flight mass spectrometry (TOF-MS). Fisher’s ratio was employed to select the key flavour compounds which could discriminate the breeding lines from the commercial cultivars. To determine whether flavour intensity is controlled by copy number of a particular gene, a “copy number” microarray was constructed by printing the genomic DNA of strawberry cultivars with different ploidy levels, ranging from haploid (from isolated microspores) to octoploid, and were hybridised with the respective genes as targets. The relationships among the five strawberry genotypes were determined by Principal Component Analysis and Hierarchical Cluster Analysis. ‘Albion’ was classified as the out group of the cluster whereas the two breeding lines, 07-102-41 and 07-095-35, were grouped together, followed by 04-069-91 and ‘Juliette’. All these data will provide useful information for DNA pooling in the subsequent Subtracted Diversity Array construction. Correlation studies will be performed between the subtractive clones and the aroma profiles to deduce the most promising DNA sequences corresponding to key flavour compounds.