A web-based software tool that enables a researcher to select optimal oligos for PCR applications and multiplex detection platforms including oligonucleotide microarrays and bead-based arrays. Given two groups of nucleic-acid sequences, a target group and a non-target group, the software identifies oligo sequences that occur in members of the target group, but not in the non-target group. To help predict potential cross hybridization, PROBEmer computes all near neighbors in the non-target group and displays their alignments. Because it does not rely upon multi-alignments, it can be applied to sequences which are not similar (e.g. genes in a single organism) or to variations of a gene in many organisms (e.g. 16S rRNA gene).