The generation of microarray probes with specificity below the species level is an ongoing challenge, not least because the high-throughput detection of microorganisms would be an efficient means of identifying environmentally relevant microbes. Here, we describe how suppression subtractive hybridization (SSH) can be applied to the production of microarray probes that are useful for microbial differentiation at the subspecies level. SSH was used to initially isolate unique genomic sequences of nine Salmonella strains, and these were validated in quadruplicate by microarray analysis. The results obtained indicate that a large group of genes subtracted by SSH could serve together, as one probe, for detecting a microbial subspecies. Similarly, the whole microbial genome (not subjected to SSH) can be used as a species-specific probe. The detailed methods described herein could be used and adapted for the estimation of any cultivable bacteria from different environments.