Accurate diagnosis of animal and zoonotic diseases, such as granulocytic anaplasmosis, is crucial to estimate risk during control programs. In this study, 16S rRNA nested PCR and RFLP assay were combined to investigate the presence of Anaplasma phagocytophilum and genetically related strains (namely A. phagocytophilum-like 1 and 2) in 936 Tunisian ruminants. By using this method, A. phagocytophilum was not detected in any of the tested animals, while A. phagocytophilum-like 1 and A. phagocytophilum-like 2 were detected at variable prevalence rates in sheep, goats and cattle at coinfection rates respectively of 3.9, 2.5 and 0.5%. Sequence analysis validated RFLP data, and confirmed the co-occurrence of two potentially novel species closely related to A. phagocytophilum in Tunisian ruminants. Phylogeny indicated the presence of genetic variants shared by different ruminant species for each type of A. phagocytophilum-like strains. Results raise concern on the use and interpretation of indirect and direct tests traditionally employed for detecting pathogenic A. phagocytophilum strains in ruminants and in other vertebrates' species, and provide additional background to improve classification of bacterial species closely related to A. phagocytophilum, and to reconstruct their evolutionary history. (C) 2017 Elsevier GmbH. All rights reserved.