This paper describes an efficient software tool ProkProbePicker (PPP) for the design of arrays of 16S rRNA-targeted probes that can be used to determine the genetic affinity of unknown bacteria. Unlike most currently available applications that have been developed to design probes for a single species or bacterial grouping, PPP was designed to extract probes for all major groupings in a known phylogenetic tree. Arrays designed by PPP will allow determination of the genetic affinity of an unknown organism relative to the groups included in the array. PPP employs the Karp-Rabin algorithm for string comparisons and the A VL data structure for a balanced binary search and therefore is more efficient than other tools. As an auxiliary part of PPP, the program OrgMap was designed to help analyze hybridization data based on probes designed by PPP. To test the capability of mapping unknown organisms based on PPP-designed probes, we conducted in silico hybridization experiments on two recently identified organisms. The results showed that a PPP designed array could correctly determine the genetic affinity of these organisms to the genus level in one case and the species level in the other. It is concluded that PPP is a useful tool for the design of arrays that can determine genetic affinity of unknown organisms.