Treatment of chronic prostatitis/chronic pelvic pain syndrome is often empirical because clinical culture methods fail to detect prostate-associated pathogens in >90% of patients. Previously, we tested a variety of specific-microorganism PCRs and began a DNA sequence study after we found that 77% of prostatitis patients were PCR positive for prokaryotic rRNA-encoding DNA sequences (rDNAs) despite negative cultures using optimal techniques. In the present study, 36 rDNA clones from 23 rDNA-positive patients were sequenced. This study represents more than twice the total rDNA sequence and more than twice the number of patients in the previous study. The increased number of patients and clones sequenced allowed enhanced phylogenetic analyses and refinements in our view of rDNA species inhabiting the prostate. A continuum of related rDNAs that might be arbitrarily described as two major groups of rDNAs and several minor groups was found. Sequences termed Pros A, identified in 8 (35%) of 23 rDNA-positive patients, grouped with Aeromonas spp. in phylogenetic studies. Sequences termed Pros B, identified in 17 (74%) of 23 rDNA-positive patients, were distinct from previously reported sequences, although all were >90% similar to known gram-negative bacteria. Of the nine patients for whom multiple rDNAs were sequenced, six had biopsy specimens containing rDNAs from more than one species. Four (17%) patients had rDNAs different from those of the Pros A and Pros B groups. Of these four, one patient had rDNA similar to that of Flavobacterium spp., another had rDNA similar to that of Pseudomonas testosteroni, and two patients had rDNAs <70% similar to known rDNAs. These findings suggest that the prostate can harbor bacteria undetectable by traditional approaches. Most of these diverse sequences are not reported in environments outside the prostate. The sequence similarities suggest adaptation of limited groups of bacteria to the microenvironment of the prostate. Further studies may elucidate the relationship of prostate-associated bacteria to chronic prostatitis/chronic pelvic pain syndrome.