The present study was aimed at simplifying procedures to delineate species and identify isolates based on DNA-DNA reassociation. DNA macro-arrays harbouring genomic DNA of reference strains of several Burkholderia species were produced. Labelled genomic DNA, hybridized to such an array, allowed multiple relative pairwise comparisons. Based on the relative DNA-DNA relatedness values, a complete data matrix was constructed and the ability of the method to discriminate strains belonging to different species was assessed. This simple approach led successfully to the discrimination of Burkholderia mallei from Burkholderia pseudomallei, but also discriminated Burkholderia cepacia genomovars I and III, Burkholderia multivorans, Burkholderia pyrrocinia, Burkholderia stabilis and Burkholderia vietnamiensis. Present data showed a sufficient degree of congruence with previous DNA-DNA reassociation techniques. As part of a polyphasic taxonomic scheme, this straightforward approach is proposed to improve species definition, especially for application in the rapid screening necessary for large numbers of clinical or environmental isolates.