Dermacentor marginatus is a known vector of Rickettsia slovaca and Anaplasma ovis distributed across Eurasia. A total of 300 D. marginatus adult male ticks were collected, removed from sheep in East Azerbaijan province, northwestern Iran. The DNA of Rickettsia and Anaplasma were found in 10 (two pools) ticks by PCR amplifying parts of ompA and msp4 genes, respectively. Our study describes the first PCR detection of tickborne pathogens, R. slovaca and A. ovis, show that they are spread in D. marginatus of Iran highlight their risk for human and animal hosts. Different patterns (ornamentation) were observed. The identity of different patterns in this study was evaluated with cytb, 16S rRNA and ITS2 genes. BLAST analysis of the cytb, 16S rRNA, ITS2 nucleotide sequence was showed 99%, 98%–100% and 98% sequence identity to sequences of D. marginatus, respectively. It was found that all different patterns of this study are in fact a polymorphic species, D. marginatus. These data increase our knowledge of tick and tick-borne microbial agents in Iran.