A polyphasic study was undertaken to determine the taxonomic position of 13 Campylobacter fetus-like strains from humans (n=8) and reptiles (n=5). The results of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and genomic data from sap analysis, 16S rRNA and hsp60 sequence comparison, pulsed- field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, DNA-DNA hybridization and whole genome sequencing demonstrate that these strains are closely related to C. fetus but clearly differentiated from the present C. fetus subspecies. Therefore this unique cluster of 13 strains represents a novel subspecies within the species C. fetus, for which the name Campylobacter fetus subsp. testudinum subsp. nov. is proposed, with strain 03-427T (=ATCC BAA-2539T = LMG 27499T) as the type strain. Although this novel taxon cannot be differentiated from C. fetus subsp. fetus and C. fetus subsp. venerealis using conventional phenotypic tests, MALDI-TOF MS analysis revealed the presence of multiple phenotypic biomarkers which distinguish C. fetus subsp. testudinum from the present C. fetus subspecies.