Shellfish including oysters (Crassostrea gigas), cockles (Cerastoderma edule) and mussels (Mytilus edulis), have previously been described as an important source of thermophilic campylobacters, with the potential of causing acute bacterial gastroenteritis in humans. Previous genotyping studies employing the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) typing, based on the flagellin (flaA) gene have been unable to generate an amplicon for the urease-positive thermophilic Campylobacter (UPTC), which are the predominant taxa associated with shellfish, largely caused by sequence diversity between the UPTC group and C. jejuni. Hence the aim of this study was to develop a modified PCR-RFLP genotyping assay, employing polymorphisms within the flagellin (flaA) gene of UPTC organisms, which would now allow the successful amplification and typing of previously nontypable UPTC isolates obtained from natural marine environments. A novel primer pair (UPTC flaF/UPTC flaR) was designed based on conserved regions within the flaA gene locus of UPTC organisms to generate a 1,358 bp amplicon for all UPTC organisms tested. RFLP analysis with DdeI in combination with computational analysis of genetic relatedness using BioNumerics software demonstrated the presence of four distinct flaA genotypes, among the seven UPTC isolates. In conclusion, this study describes a PCR-RFLP method, based on modified primers from UPTC flaA gene sequences that may be successfully applied to examine subspecies relatedness of UPTC organisms from natural environments, including shellfish.