Chlamydophila abortus is an important pathogen in human and a variety of animals. Ovine chlamydiosis (also known as Enzootic Abortion of Ewes (EAE)) is a venereal transmitted disease associated with late abortion and economic impact in sheep production. There is a lack of information about its status in Palestine, in which sheep are the most abundant farm animal that has a traditional and economic importance. Particularly, the infection rate in male is not well documented. In the present study, 2608 blood samples were collected from active reproductive rams between April and June 2016 in five Palestinian cities (Jenin, Nablus, Jericho, Tulkarm, and Tubas). Sera were tested by ID Screen® Chlamydophila abortus Indirect Multi-species enzyme-linked immunosorbent assay (ELISA) kit for detection of C. abortus immunoglobulin G (IgG) (IDvet, France). At the individual level, among 2608 samples analyzed, 385 (13.7%) were seropositive for C. abortus. The chi-square analysis was used to evaluate the risk factors associated with regions, which could provide the foundation to understand the occurrence of the diseases. There was a significant difference between the regions studied (P <0.05). Our study revealed C. abortus seroprevalence rate of 17.7% in Nablus and Tubas cities, followed by Jericho (14.2%), Jenin (9.6%) and Tubas (3.8%). At the herd level, among the 353 farms studied, 188 (53.3 %) had at least one seropositive animal. The highest rate was found in the herds of Tubas city (90.9%), followed by Nablus (78.3%), Jenin (56.9%), Jericho (44.9%) and Tulkarm (36.4%). Most of the seropositive cases were observed in cities with large herd size. In conclusion, detection of C. abortus antibody in rams serum suggests a potential for venereal transmission that further attention should be paid to this disease in this region of the country.