The aims of this study consist of first estimating the molecular prevalence of Anaplasma ovis with specific species using the loop-mediated isothermal amplification (LAMP) method in sheep (n = 204) from Northern and Central Tunisia. This study also characterizes the genotypes of this Anaplasma species by partial msp4 gene sequence. The molecular prevalence of A. ovis used to record 70.1%. Prevalence rate was significantly higher in El Alia locality (governorate of Bizerte, Northern Tunisia), where it indicated 91.1%, than in Sbikha locality (governorate of Kairouan, Central Tunisia), where it indicated 49.5%. In Sbikha, A. ovis prevalence varied significantly according to two factors, ie, gender and age. In fact, ewes were significantly more infected than ram lambs. Ticks collected on animals allowed recovering 271 ixodid ticks from El Alia. Ticks belonged to 3 species of Rhipicephalus genus: first, R. turanicus (59.78%) which was the dominant species and which was followed by R. sanguineus (36.16%) and R. annulatus (4.06%). In Sbikha, an absence of ticks was noted in all of the examined sheep. The analysis of A. ovis msp4 sequences revealed four different genotypes; two of them were previously described in Italy and two new others genotypes were classified in an independent cluster. The present work is the first published report of A. ovis infection in sheep in Tunisia. The results indicate the presence of A. ovis at a high rate; hence, the need for a survey into the medical and economic impact of the disease, and the identification of vectors of this bacterium in Tunisia must be made.