Differentiation of Campylobacter fetus into C. fetus subsp. fetus (Cff) and C. fetus subsp. venerealis (Cfv) is important for both clinical and economic reasons. In the past, several molecular typing methods have been used for differentiation, including amplified fragment length polymorphism (AFLP). In this study, AFLP was employed to identify C. fetus subspecies specific markers that can serve as a basis for design of novel PCR primer sets for Cfv. Four groups of C. fetus strains with different phenotypic or genotypic traits were examined by AFLP using 22 different DdeI/MboI primer combinations. Specific AFLP fragments were deduced and sequenced resulting in 41 sequences. Based on the obtained sequences, five potential subspecies-specific PCR assays were developed. Extensive evaluation of the five selected PCRs with a set of 65 diverse C. fetus strains identified primer set Cf C05 as subspecies Cfv-specific. This newly developed PCR is fully consistent with the AFLP subspecies differentiation results. The data indicate AFLP as a powerful tool for comparing closely related genomes and for exploiting this information to develop a specific PCR with extensive typing potential.