A panel of three DNA probes were derived at random from a genomic DNA library of Campylobacter mucosalis strain E8384-4. Each probe hybridized specifically to C. mucosalis DNA from bacteria fixed to nylon membranes. The probes did not hybridized to DNA from other Campylobacter species or to other bacteria even at 100-fold higher amounts. Each probe hybridized to all of 24 isolates of C. mucosalis which had been collected over time from different geographic locations. Southern blot analysis of selected C. mucosalis isolates was carried out to determine if the probes would be useful for differentiating among various isolates. It indicated that restriction fragment length polymorphisms (RFLPs) exist at the loci identified by our probes. These differences were used to characterize seven C. mucosalis isolated recovered from pigs in Minnesota. The results suggest that RFLP analysis may be useful tool for epidemiological studies of C. mucosalis.